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- Complexin arrests a neighbor
- Nat Struct Mol Biol 18(8):861-863 (2011)
Article preview View full access options Nature Structural & Molecular Biology | News and Views Complexin arrests a neighbor * Keith R Weninger1Journal name:Nature Structural & Molecular BiologyVolume: 18,Pages:861–863Year published:(2011)DOI:doi:10.1038/nsmb.2118Published online03 August 2011 Mechanistic details about complexin's contradictory double life as both a facilitator and an inhibitor of SNARE-mediated synaptic vesicle fusion have been challenging to uncover. A series of studies in this issue addresses the problem by revealing a switchable complexin conformation in which fusion arrest occurs when complexin clamps neighboring SNAREs. Figures at a glance * Figure 1: Complexin modulates SNARE-driven membrane fusion of synaptic vesicles. Left, trans-SNARE complexes assembled from t-SNAREs (syntaxin, yellow; SNAP-25, green) and a v-SNARE (synaptobrevin, blue) dock synaptic vesicles to the presynaptic plasma membrane. Synaptotagmin (gray ovals) and complexin (cyan) are shown interacting with the SNAREs, as seen in structural models10, 19. Right, transitions to the state with an open fusion pore. Below is a schematic illustrating the four major domains of complexin, with approximate amino acid numbers marking domain boundaries. * Figure 2: Schematics illustrating the configuration of complexin. (,) The accessory helix (cyan) is bent away from the trans-SNARE complex () and cross-linked into a zigzag array (), as determined by Kümmel et al.2 () Transitions necessary for opening a fusion pore. The v-SNARE shown in (blue) is truncated, and the trans-SNARE bundle and complex (red) shown in and , respectively, have been simplified. 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 * Keith R. Weninger is in the Department of Physics, North Carolina State University, Raleigh, North Carolina, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Keith R Weninger Author Details * Keith R Weninger Contact Keith R Weninger Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Conformational flexibility and rotation of the RING domain in activation of cullin–RING ligases
- Nat Struct Mol Biol 18(8):863-865 (2011)
Article preview View full access options Nature Structural & Molecular Biology | News and Views Conformational flexibility and rotation of the RING domain in activation of cullin–RING ligases * Simin Rahighi1 * Ivan Dikic2 * Affiliations * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:863–865Year published:(2011)DOI:doi:10.1038/nsmb.2117Published online03 August 2011 The RING protein RBX-1 is implicated in both NEDDylation and ubiquitylation reactions. In this issue, new structural analysis reveals how conformational flexibility of the RBX-1 linker allows for a marked reorientation of the CUL1–RBX1 complex to facilitate transfer of NEDD8 or ubiquitin by closing the gap between the E2 catalytic site and the substrate. 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 * Simin Rahighi is at the Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Japan. * Ivan Dikic is at the Frankfurt Institute for Molecular Life Sciences, Institute of Biochemistry 2, Goethe University School of Medicine, Frankfurt, Germany. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ivan Dikic Author Details * Simin Rahighi Search for this author in: * NPG journals * PubMed * Google Scholar * Ivan Dikic Contact Ivan Dikic Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - A bottle opener for TBP
- Nat Struct Mol Biol 18(8):865 (2011)
Article preview View full access options Nature Structural & Molecular Biology | News and Views A bottle opener for TBP * Sabbi LallJournal name:Nature Structural & Molecular BiologyVolume: 18,Page:865Year published:(2011)DOI:doi:10.1038/nsmb0811-865Published online03 August 2011 The ATP-dependent remodelers are implicated in many DNA transactions. Although considerable mechanistic insight has been gained through biochemical, biophysical and in vivo analyses, structural information that shows how these complexes work to move proteins relative to DNA has been elusive. Auble, Hopfner and colleagues (Nature doi:10.1038/nature10215, advance online publication 6 July 2011) have now examined the structure of Mot1, an ATPase in the Swi2/Snf2 family that acts as a single polypeptide. The fact that Mot1 can act alone makes it a good candidate for structural analysis, given that a number of ATP-dependent chromatin remodelers in this family act as members of large complexes. Mot1 is highly conserved in the eukaryotes and is a regulator of TATA box–binding protein (TBP). Because it quite specifically displaces TBP from DNA, it has a simple substrate facilitating analysis, and it has an interesting role in transcriptional regulation. The authors have solved the! structure of the N-terminal domain (NTD) of Encephalitozoon cuniculi Mot1 in complex with EcTBP to 3.1-Å resolution. This region has previously been implicated in TBP binding and lacks the ATPase that is located in the Mot1 C-terminal region. The authors found that the NTD of Mot1 consists of 16 HEAT repeats that are organized in a horseshoe shape (yellow and orange in image). The Mot1 repeats wrap around a single TBP molecule (blue in image), of interest because TBP can also form dimers. Mot1 interacts across the convex side of TBP, a region known to interact with other proteins. However, Mot1 also unexpectedly wraps around and interacts with the DNA-binding surface of TBP, immediately suggesting a mechanism by which it might prevent TBP interaction with DNA. The authors called this region of Mot1 the 'latch' (magenta in image). The interactions observed in the structure overlap with some seen in the TBP–TBP dimer and the TBP–DNA structure. Indeed, TBP adopts a very ! similar conformation in all of these structures. Mutational analysis in the literature, both in vivo and in vitro, as well as further analysis presented by the authors support the concept of Mot1 interactions across the convex back of TBP. Indeed, a broad set of interactions in the N-terminal domain of BTAF1 (the human ortholog of Mot1) and Saccharomyces cerevisiae Mot1 has previously been implicated in TBP interactions, consistent with the present structure. In order to test the role of the latch region in TBP interactions, the authors generated deletion mutants of the full-length and Mot1 (NTD) proteins that lack latch residues. As expected, these deletion mutants did in fact interact with TBP, given that they contain the HEAT repeats required for interaction across the convex side of TBP; however, in this case, the NTD of Mot1 without the latch interacted with a TBP dimer. This supports the idea that the latch may be involved in disrupting the TBP–TBP dimer. The latch mutant Mot1 (NTD) also inhibited DNA binding in vi! tro when incubated with TBP prior to DNA exposure. It was not, however, absolutely required for inhibiting the TBP-DNA interaction, based on experiments with the Mot1 latch mutant containing the C-terminal ATPase domain. This suggests a model in which the ATPase region can displace TBP from DNA, and the latch facilitates or makes this more efficient, perhaps by interacting with the hydrophobic face of TBP and preventing DNA rebinding. 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. Additional data - Research highlights
- Nat Struct Mol Biol 18(8):866 (2011)
Article preview View full access options Nature Structural & Molecular Biology | Research Highlights Research highlights * Inês Chen * Arianne Heinrichs * Sabbi Lall * Steve MasonJournal name:Nature Structural & Molecular BiologyVolume: 18,Page:866Year published:(2011)DOI:doi:10.1038/nsmb0811-866Published online03 August 2011 Anchored tails Tail-anchored (TA) membrane proteins contain a C-terminal membrane anchor and are post-translationally delivered to the ER by the Get3 ATPase. Get3 interacts with the two receptor proteins Get1 and Get2 at the membrane, but the molecular mechanism underlying Get3-dependent membrane insertion of TA proteins has been unknown. Denic, Dötsch, Sinning and colleagues now report the crystal structures of Get3 in complex with the cytosolic domains of Get1 and Get2 in different functional states. Both receptor proteins are required for TA proteins to insert in a nucleotide-dependent manner. The cytosolic domain of Get1, which is necessary and sufficient for Get3 binding, was crystallized in complex with Get3. The Get3–Get1 complex is a symmetric heterotetramer, with two Get3 molecules in the open state and two Get1 molecules binding at the interface of the Get3 dimer. Get1 contacts both Get3 monomers, with one interaction surface providing high affinity while the second interferes! with nucleotide binding and forms only with the open state of Get3. The authors also determined the structures of the semi-open Get3–Get1 complex and the closed Get3–Get2 complex. Comparing the two Get3–Get1 structures suggests that Get1 stays bound to Get3 through the high-affinity interface during the transition from the fully closed to the open state. In contrast to Get1, two Get2 molecules bind away from the Get3 dimer interface such that each Get2 molecule contacts only one Get3 subunit. Get1 and Get2, which can bind simultaneously to Get3, share a partially overlapping binding site. The authors propose that TA protein binding locks Get3 in the closed state and that Get2 tethers Get3–TA to the ER membrane. Get1 associates with Get3–TA–Get2, partially displacing Get2 and resulting in an insertion-competent complex. Although the precise timing of ATP hydrolysis is still unknown, the authors favor a scenario in which TA protein binding induces ATP hydrolysis ! in Get3. Upon interaction with the Get1-Get2 receptor, the sto! red energy from hydrolysis drives Get3–Get1 through the semi-open to the open state and facilitates TA protein insertion. (Science doi:10.1126/science.1207125, published online 30 June 2011) AH Small but mighty Small molecules have been used to target specific proteins and regulate their activity in vivo. Two recent papers describe systems in which small molecules control the levels of target proteins by triggering their degradation. The first report is from Wandless and colleagues, who previously developed systems fusing the protein of interest to destabilizing domains that cause its rapid degradation. One such system used FK506- and rapamycin-binding protein (FKBP) as the destabilizing domain; the small molecule Shield-1 binds and stabilizes FKBP. Now these authors turn the concept around and engineer a version of FKBP with a C-terminal 19-residue tail, which binds to FKBP's active site and stabilizes it. When Shield-1 is added, the C-terminal tail is displaced and acts as a degron to promote rapid degradation of the fusion; a four-residue motif (RRRK) was shown to be sufficient to cause rapid degradation of yellow fluorescent protein (YFP). In the second paper, Crews and colleag! ues use the commercial HaloTag system, in which the protein of interest is fused to a modified bacterial dehalogenase that can form a covalent bond with synthetic bifunctional ligands. The authors screened HaloTag ligands with different hydrophobic moieties for their ability to destabilize fusion proteins. The rationale was that the fusion protein containing such an exposed hydrophobic tag would be recognized as an unfolded protein and eliminated through the ubiquitin-proteasome system. The selected hydrophobic tag was shown to work well in cell culture and in animal models (zebrafish embryos and mice). Both systems are a welcome addition to the arsenal of tools to manipulate the levels of proteins of interest in cells, in a rapid and efficient way. (Nat. Chem. Biol. doi:10.1038/nchembio.598 and doi:10.1038/nchembio.597, published online 3 July 2011) IC 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 Author Details * Inês Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Arianne Heinrichs Search for this author in: * NPG journals * PubMed * Google Scholar * Sabbi Lall Search for this author in: * NPG journals * PubMed * Google Scholar * Steve Mason Search for this author in: * NPG journals * PubMed * Google Scholar - Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition
- Nat Struct Mol Biol 18(8):867-874 (2011)
Nature Structural & Molecular Biology | Article Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition * Oliver G McDonald1, 2, 3 * Hao Wu4 * Winston Timp1, 2 * Akiko Doi1, 2 * Andrew P Feinberg1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:867–874Year published:(2011)DOI:doi:10.1038/nsmb.2084Received30 December 2010Accepted04 May 2010Published online03 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 Epithelial-to-mesenchymal transition (EMT) is an extreme example of cell plasticity that is important for normal development, injury repair and malignant progression. Widespread epigenetic reprogramming occurs during stem cell differentiation and malignant transformation, but EMT-related epigenetic reprogramming is poorly understood. Here we investigated epigenetic modifications during EMT mediated by transforming growth factor beta. Although DNA methylation was unchanged during EMT, we found a global reduction in the heterochromatin mark H3 Lys9 dimethylation (H3K9Me2), an increase in the euchromatin mark H3 Lys4 trimethylation (H3K4Me3) and an increase in the transcriptional mark H3 Lys36 trimethylation (H3K36Me3). These changes depended largely on lysine-specific demethylase-1 (Lsd1), and loss of Lsd1 function had marked effects on EMT-driven cell migration and chemoresistance. Genome-scale mapping showed that chromatin changes were mainly specific to large organized hete! rochromatin K9 modifications (LOCKs), which suggests that EMT is characterized by reprogramming of specific chromatin domains across the genome. View full text Figures at a glance * Figure 1: Global changes in bulk chromatin modifications during EMT. () Hematoxylin and eosin (H&E) stains of differentiated AML12 cells and those treated with TGF-β, highlighting stellate morphology, nuclear enlargement and altered chromatin texture during EMT. Yellow scale bars represent 20 μm for 40× panels and 80 μm for 10× panels. () Electron microscopy of differentiated AML12 cells and those treated with TGF-β, highlighting smooth chromatin, scattered nucleoli and loss of peripheral heterochromatin during EMT. Yellow scale bars represent 1 μm for 25,000× panels and 5 μm for 5,000× panels. () Changes in bulk chromatin modifications during EMT. Representative western blots for the indicated histone H3 modifications of bulk histones from AML12 cells treated with TGF-β for the indicated time points. 'TGF-β on' refers to time points during which cells were treated with TGF-β, whereas 'TGF-β off' refers to time points after which TGF-β was removed from the medium, allowing termination of EMT and re-differentiation to hepatocyte! s (as in Supplementary Fig. 1). There was a reversible reduction in H3K9Me2 and increase in H3K4Me3 and H3K36Me3 in response to TGF-β. Total levels of histone H3 remained relatively constant. () Densitometry quantification summarizing western blots of histone modifications (including those depicted in ; n = 2–3 biological replicates) for indicated times of TGF-β treatments. Data are plotted as raw signal intensities. Error bars represent s.e.m. () Western blots of whole-cell extracts from AML12 cells treated with siRNA against E-cadherin (siEcad) or control siRNA against GFP (siGFP) show reduced E-cadherin and increased vimentin in response to siEcad, consistent with induction of EMT. () Changes in bulk chromatin modifications in response to siEcad. Representative western blots for the indicated H3 modifications of bulk histones treated as indicated show similar changes as seen in . () Densitometry quantification summarizing western blots of histone modifications (inclu! ding those depicted in ; n = 2 biological replicates) for indi! cated siRNA treatments. Data are plotted as raw signal intensities. Error bars represent s.e.m. * Figure 2: Lsd1 regulates bulk changes in chromatin modifications during EMT. () Western blot of whole-cell extracts from AML12 cells treated with TGF-β for the indicated time points (as in Fig. 1). Lsd1 expression was increased by TGF-β treatment. () Venn diagram summarizing proteins that preferentially immunoprecipitated with Lsd1 from differentiated AML12 cells (vehicle) and those undergoing EMT (TGF-β). Proteins were identified by large-scale immunoprecipitation of endogenous Lsd1 from nuclear extracts followed by mass spectrometry of immunoprecipitates that had been separated on a polyacrylamide gel. 'Both' refers to proteins that immunoprecipitated with Lsd1 under both conditions. We detected several known Lsd1 interactions during both conditions (for example, BHC80, CTBP1 and HMG20A). By contrast, other interactions were specific for vehicle-treated cells, including proteins involved in heterochromatin assembly (for example, CoREST, STAT3 and UBXD2). TGF-β treated cells yielded several proteins that are important for regulation of chromatin! , EMT and oncogenesis (for example, several catenins, DEAD box RNA helicases, Rac1 and PARP1). () Western blots of the indicated proteins from AML12 cells treated with or without (vehicle-only) TGF-β, and incubated with the siRNAs listed in the panel above the blots. Plus denotes cells that received the indicated treatments, minus denotes cells that did not. siRNAs against Lsd1 but not GFP (control) knocked down Lsd1 expression but did not prevent upstream events that initiate EMT (loss of E-cadherin, gain of vimentin), as shown by western blot. () Western blots of H3K9Me2, H3K36Me3 and H3K4Me3 (as in ). siLsd1 prevented loss of H3K9Me2 and partially interfered with gain of H3K36Me3 and H3K4Me3 in response to TGF-β. Total levels of histone H3 remained constant. () Densitometry quantification plots summarizing western blot data for the indicated siRNA treatments (including those shown in , n = 2 biological replicates). Data are plotted as raw signal intensities. Error bars! represent s.e.m. () Pargyline treatment does not affect E-cad! herin, vimentin or Lsd1. AML12 cells were incubated with TGF-β alone or with both TGF-β (TGF) and pargyline (Pg) as indicated. Pargyline did not prevent the induction of upstream aspects of EMT (E-cadherin, vimentin) and did not affect expression of Lsd1, as shown by western blots. () Pargyline treatment blocked chromatin changes during EMT. Pargyline prevented the loss of bulk H3K9Me2 and gain of H3K36Me3 in response to TGF-β, and total levels of histone H3 remained relatively constant. H3K4Me3 levels rose even further. () Densitometry quantification summarizing western blots for pargyline experiments (including those depicted in , n = 2 biological replicates). Data are plotted as raw signal intensities. Error bars represent s.e.m. * Figure 3: Lsd1 regulates certain EMT phenotypes. () Light microscopy of representative areas of scratch assays on cultures of AML12 cells treated as indicated. siLsd1 partially inhibited cell migration as shown by reduced numbers of cells that have migrated into the scratched area. siLsd1 caused increased migration in vehicle-treated cells. Black scale bars represent 40 μm. () Quantification of representative scratch assays (n = 2) as above. Cells in the scratch area were counted over ten microscopic fields for each condition. The average numbers of migratory cells are plotted; error bars represent s.d. siLsd1 partially blocked cell migration in TGF-β–treated cells. () Light microscopy of representative areas of scratch assays performed on cultures of AML12 cells treated as indicated. Plus and minus signs together refers to cells that were treated with TGF-β and pargyline (Pg) for 36 h, after which pargyline was removed from the medium for 36 h. Pargyline inhibited cell migration as shown by the lack of cells that hav! e migrated into the scratched area. Black scale bars represent 40 μm. () Quantification of representative scratch assays as in . Individual cells in the scratch area were counted over ten microscopic fields for each condition. The average numbers of migratory cells are plotted; error bars represent s.d. Pargyline completely blocked cell migration. () Representative dose-response curves of AML12 cells treated as indicated and exposed to increasing concentrations of doxorubicin. Plots are absorbance values normalized to no drug (DMSO only) and indicate cell viability. siLsd1 abolished resistance to doxorubicin (n = 2, error bars represent s.d.; *P < 0.05 relative to vehicle + siGFP controls). () Representative dose-response curves of AML12 cells treated as indicated and exposed to increasing concentrations of doxorubicin. Plots are absorbance values normalized to no drug (DMSO only), and indicate cell viability. Pargyline conferred enhanced resistance to doxorubicin (n = 2, ! error bars represent s.d.;*P < 0.05 relative to vehicle contro! ls). * Figure 4: ChIP-chip analysis of EMT reveals alterations of chromatin in LOCK domains. (–) Representative plots of ChIP-chip enrichments of histone modifications over loci from mouse chromosomes 12 (), 10 () and 4 () during TGF-β–induced EMT. Locations of genes that were assayed by RT-PCR are shown at the bottom. Regions where we detected LOCKs (top, H3K9Me2), K4Me3 LOCKs (middle, H3K4Me3) and peaks of H3K36Me3 (bottom, H3K36Me3) are shown as gray bars. Chromatin was harvested from differentiated AML12 cells (time 0 h) and from AML12 cells undergoing EMT (TGF-β–treated, 36 h). H3K9Me2 was reduced and H3K4Me3 was enriched in LOCK regions. Areas with enriched H3K36Me3 are present over genes at the boundaries of these LOCKs. See Supplementary Figures 8,9 for replicates of various regions, including region shown in by ChIP-qPCR. Graphs underneath the ChIP-chip plots show RT-PCR results for the genes beneath them, showing that genes at LOCK boundaries that acquire H3K36Me3 are upregulated during EMT. () Histogram comparing GC content of LOCKs, K4Me3 LOCKs a! nd the whole genome. LOCKs are AT-rich overall but those that acquire H3K4Me3 during EMT are GC rich. () Gene content in all LOCKs, K4Me3 LOCKs and the whole genome. LOCKs are gene poor overall, but the subset of LOCKs that acquire K4Me3 during EMT are gene enriched over LOCKs that do not. () ChIP assays for Lsd1 across the locus depicted in . ChIP assays were performed for Lsd1 and quantitative real-time PCR was performed with 31 primers spaced across the locus. Gray bars represent K4Me3 LOCKs and H3K36Me3-enriched boundaries. Whereas Lsd1 binding was restricted to the 5′ end of the Lsd1 and Epha8 genes in differentiated cells, Lsd1 is enriched across the entire K4Me3 LOCK during EMT. () ChIP assays for Lsd1 as in show that chromatin isolated from AML12 cells treated with both TGF-β and siLsd1 shows loss of Lsd1 enrichment within the K4Me3 LOCK during EMT. () ChIP assays for Lsd1 as in show that chromatin isolated from AML12 cells co-treated with TGF-β and pargyline sh! ows retention of Lsd1 enrichment within the K4Me3 LOCK during ! EMT. () Model of LOCK reprogramming during EMT. In differentiated AML12 cells, LOCKs have high levels of H3K9Me2. Lsd1 forms complexes with proteins that might facilitate heterochromatin assembly at LOCKs. During TGF-β–mediated EMT, Lsd1 spreads from LOCK boundaries into the LOCKs and other proteins converge upon Lsd1, which may assist in directing demethylation of H3K9Me2 and recruitment of H3K4Me3. H3K9Me2 in all LOCKs is reduced, and specific, GC-rich LOCKs acquire H3K4Me3 adjacent to sites of transcription. These K4Me3 LOCKs might function in a surveillance DDR pathway, providing enhanced chemoresistance during EMT. H3K36Me3 is targeted to K4Me3 LOCK boundaries and numerous EMT-related genes in non-LOCK regions across the genome, which are involved in conferring cell motility during EMT. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE28581 * GSE27968 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Center for Epigenetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Oliver G McDonald, * Winston Timp, * Akiko Doi & * Andrew P Feinberg * Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Oliver G McDonald, * Winston Timp, * Akiko Doi & * Andrew P Feinberg * Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Oliver G McDonald * Department of Biostatistics and Bioinformatics, Emory University, Atlanta, Georgia, USA. * Hao Wu Contributions O.G.M. and A.P.F. conceived this work, designed the experiments and wrote the manuscript. H.W. performed the statistical analyses. W.T. performed the immunofluorescence. A.D. performed the CHARM. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Andrew P Feinberg Author Details * Oliver G McDonald Search for this author in: * NPG journals * PubMed * Google Scholar * Hao Wu Search for this author in: * NPG journals * PubMed * Google Scholar * Winston Timp Search for this author in: * NPG journals * PubMed * Google Scholar * Akiko Doi Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew P Feinberg Contact Andrew P Feinberg Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information Excel files * Supplementary Data 1 (188K) Locations of K9Me2 LOCKs identified in AML12 cells PDF files * Supplementary Text and Figures (2.9M) Supplementary Figures 1–9 and Supplementary Tables 1–5 Additional data Entities in this article * REST corepressor 1 Rcor1 Mus musculus * View in UniProt * View in Entrez Gene * RE1-silencing transcription factor Rest Mus musculus * View in UniProt * View in Entrez Gene * Zinc finger protein SNAI1 Snai1 Mus musculus * View in UniProt * View in Entrez Gene * Ephrin type-A receptor 8 Epha8 Mus musculus * View in UniProt * View in Entrez Gene * Vimentin Vim Mus musculus * View in UniProt * View in Entrez Gene * Cadherin-1 Cdh1 Mus musculus * View in UniProt * View in Entrez Gene * PHD finger protein 21A Phf21a Mus musculus * View in UniProt * View in Entrez Gene * Ki-67 protein Mki67 Mus musculus * View in UniProt * View in Entrez Gene * 60S acidic ribosomal protein P1 Rplp1 Mus musculus * View in UniProt * View in Entrez Gene * Ras-related protein Rab-30 Rab30 Mus musculus * View in UniProt * View in Entrez Gene * Histone H2A.x H2afx Mus musculus * View in UniProt * View in Entrez Gene * Lysine-specific histone demethylase 1A Kdm1a Mus musculus * View in UniProt * View in Entrez Gene * UBX domain-containing protein 4 Ubxn4 Mus musculus * View in UniProt * View in Entrez Gene * Signal transducer and activator of transcription 3 Stat3 Mus musculus * View in UniProt * View in Entrez Gene * High mobility group protein 20A Hmg20a Mus musculus * View in UniProt * View in Entrez Gene * C-terminal-binding protein 1 Ctbp1 Mus musculus * View in UniProt * View in Entrez Gene * Poly [ADP-ribose] polymerase 1 Parp1 Mus musculus * View in UniProt * View in Entrez Gene * Ras-related C3 botulinum toxin substrate 1 Rac1 Mus musculus * View in UniProt * View in Entrez Gene - Crystal structure of a chaperone-bound assembly intermediate of form I Rubisco
- Nat Struct Mol Biol 18(8):875-880 (2011)
Nature Structural & Molecular Biology | Article Crystal structure of a chaperone-bound assembly intermediate of form I Rubisco * Andreas Bracher1, 2 * Amanda Starling-Windhof1, 2 * F Ulrich Hartl1 * Manajit Hayer-Hartl1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:875–880Year published:(2011)DOI:doi:10.1038/nsmb.2090Received04 March 2011Accepted20 May 2011Published online17 July 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 The form I Rubisco of autotrophic bacteria, algae and plants is a complex of eight large (RbcL) and eight small (RbcS) subunits. It fixes atmospheric CO2 in the dark reaction of photosynthesis. As shown for the cyanobacterial enzyme, folding of the RbcL subunits is mediated by the GroEL–GroES chaperonin system, and assembly requires the specialized chaperone RbcX, a homodimer of ~15-kDa subunits. Here we present the 3.2-Å crystal structure of a Rubisco assembly intermediate, consisting of the RbcL8 core with eight RbcX2 molecules bound. The structure reveals the molecular mechanism by which RbcX2 mediates oligomeric assembly. Specifically, RbcX2 provides positional information for proper formation of antiparallel RbcL dimers, thereby preventing RbcL–RbcL misalignment and off-pathway aggregation. The RbcL8(RbcX2)8 structure also suggests that RbcS functions by stabilizing the '60s loop' of RbcL in the catalytically active conformation. View full text Figures at a glance * Figure 1: Overall architecture and dimensions of the RbcL8(RbcX2)8 complex. The RbcL8 core structure of Syn6301 is shown in surface representation and the bound RbcX dimers in ribbon representation. Colors indicate individual peptide chains: alternating RbcL subunits are in yellow and orange, and the RbcX chains in green and light green, respectively. * Figure 2: The interactions of RbcX2 and RbcL in molecular detail. () Close-up view showing surfaces on the antiparallel dimer of RbcL that interact with RbcX2. The outline of the bound RbcX2 is shown for orientation. The interaction surfaces area 1 (purple) and area II (cyan) are located on one RbcL subunit, whereas area III (red) is located on the adjacent subunit of the RbcL dimer. () Ribbon diagram of the RbcX dimer showing the contact regions with RbcL, colored as in . RbcX2 is rotated 180° relative to the view shown in . () Close-up view of the RbcX2 interface with the C-terminal peptide of RbcL (area I). RbcX2 is shown in surface representation; interface residues are indicated. The C-terminal peptide of RbcL is shown in stick model. In the background, the area II contact between loop 6 of RbcL and residue Gln5 of one RbcX chain (green) is visible. Oxygen and nitrogen heteroatoms are indicated in red and blue, respectively. () Cutaway view of the RbcX2 interface with the opposing RbcL subunit (area III). The surface of RbcX2 is show! n as a transparent skin. Crucial contact residues in RbcX and RbcL are shown in stick representation. The β-sheet in the N-terminal domain of RbcL, which would be in the foreground, is omitted for clarity. * Figure 3: Probing the interaction of RbcX2 and RbcL by mutagenesis of the heterologous and cognate complex. () Analysis of mutants in the RbcL N-terminal domain. Left, a close-up view of the RbcL N-terminal domain showing the interface area III for RbcX2 binding. The mutated residues Glu49, Ala53 and Ala126 are indicated. Right, native PAGE analysis of AnaCA-RbcX2 binding to RbcL8 complexes of wild-type (WT-RbcL) and mutant RbcL. Note that high-affinity binding of AnaCA-RbcX2 to WT-RbcL8 results in an upshift of the complex (lane 2) (see Online Methods). () Analysis of the RbcL-RbcX2 interface via designed disulfide bonds. Left, design of a RbcL–RbcX disulfide bond at the area III interface. RbcX2 is shown as a transparent surface and RbcL in ribbon representation. The positions of the cysteine mutations and the disulfide linkage are modeled for the RbcL8 complex with AnaCA-RbcX2. Right, disulfide bond formation between RbcL and AnaCA-RbcX (lane 3) or Syn6301-RbcX (lane 7) upon coexpression of RbcL and RbcX proteins and incubation of the isolated complexes under oxidizing condit! ions (see Online Methods). Reactions were analyzed by nonreducing SDS-PAGE and immunoblotting. * Figure 4: Role of RbcX2 in forming and stabilizing the antiparallel RbcL dimer. () Distribution of hydrophobic and charged surface residues at the dimer interface. RbcL is shown in surface representation and the adjacent subunit is outlined. Positively and negatively charged functional groups are shown in blue and red, respectively, hydrophobic side chains in yellow and the rest of the surface in white. One bound RbcX2 is shown in ribbon representation and residue Arg69 in space-filling representation. () RbcL dimer interface is shown as a contact residue footprint (white) on one RbcL subunit, oriented as in . () Electrostatic potential map of RbcL. The electrostatic potential of an RbcL subunit was calculated and mapped onto its van der Waals surface. A red-white-blue color gradient indicates surface potential values from −25 to +25 kBT. () Electrostatic potential map of the RbcL–RbcX2 complex. Bound RbcX2 extends the interface to the opposing RbcL subunit. () Rescue of dimer assembly of RbcL mutants by RbcX2. RbcL mutants E106Q and R212S were eith! er expressed alone or coexpressed with AnaCA-RbcX2. Soluble cell lysates were analyzed by SDS-PAGE and immunoblotting to detect total soluble RbcL and by native PAGE and immunoblotting to detect RbcL assembly intermediates. Wild-type RbcL (WT-RbcL) was used as control. () Interface between RbcL dimers indicating the position of residue Arg212. * Figure 5: Comparison of RbcL in complex with RbcX2 and in the CABP-bound Rubisco holoenzyme6. () RbcL in complex with RbcX2. One RbcL2(RbcX2)2 unit of the RbcL8(RbcX2)8 complex is shown. RbcL subunits and RbcX2 are depicted in ribbon and surface representations, respectively. Disordered regions are indicated by dots. () RbcL in the CABP-bound Rubisco holoenzyme. The RbcL chains are colored gray and cyan; RbcS is colored purple. CABP is shown as space-filling model in red. () Superposition of RbcL8(RbcX2)8 and the holoenzyme complex. Note the steric clash with RbcX2, which would occur when the 60s loop is structured as in the holoenzyme. Arrows indicate the direction of displacement of the RbcL subunits when RbcX2 is bound. () Role of the 60s loop in RbcS-mediated displacement of RbcX2. Left, close-up view of the interface of RbcS and the 60s loop with residues Trp67, Leu70 and Leu71 of the 60s loop shown in space-filling representation in green. Right, displacement by purified RbcS (0–4 μM) of wild-type RbcL (WT-RbcL) or RbcL(W67A L70G L71A) triple mutant from imm! obilized RbcL8(Syn6301-RbcX2)8Flag complex (see Online Methods). RbcL protein that remained bound was detected by SDS-PAGE and immunoblotting. () Ability of RbcL(W67A L70G L71A) to form the RbcL8S8 complex (enzymatically inactive). Soluble cell lysates containing WT-RbcL or RbcL(W67A L70G L71A) were analyzed by native PAGE and immunoblotting in the absence or presence of purified RbcS (3 μM). * Figure 6: Model for RbcX2-assisted Rubisco assembly. Upon folding and release from the GroEL–GroES complex, the RbcL subunit is recognized by RbcX2, which binds the flexible C-terminal RbcL peptide (area I) as well as area II on the folded body of the subunit (step 1). The antiparallel RbcL dimer then forms (step 2); the complementary surface charges on both RbcL and RbcX2 are likely to have a role in guiding dimer formation, avoiding misalignment. The RbcX2 molecules, each contacting area III on the respective adjacent RbcL subunit, function as 'molecular staples' in stabilizing the dimer. The stable RbcL2(RbcX2)2 units subsequently assemble to the RbcL8 core complex (step 3). In this complex, a large portion of the RbcS-binding interface is preformed. RbcS binding structures the RbcL N terminus and the 60s loop, which sterically blocks access of RbcX2 to binding area III on RbcL. This facilitates displacement of RbcX2 and formation of the functional Rubisco holoenzyme (step 4). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Andreas Bracher & * Amanda Starling-Windhof Affiliations * Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany. * Andreas Bracher, * Amanda Starling-Windhof, * F Ulrich Hartl & * Manajit Hayer-Hartl Contributions A.B. crystallized the Rubisco assembly intermediate, solved the structure and designed the mutants. A.S.-W. executed the biochemical study. A.B., A.S.-W., F.U.H. and M.H.-H. contributed to experimental design, analysis and interpretation of data. A.B., F.U.H. and M.H.-H. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Andreas Bracher or * Manajit Hayer-Hartl Author Details * Andreas Bracher Contact Andreas Bracher Search for this author in: * NPG journals * PubMed * Google Scholar * Amanda Starling-Windhof Search for this author in: * NPG journals * PubMed * Google Scholar * F Ulrich Hartl Search for this author in: * NPG journals * PubMed * Google Scholar * Manajit Hayer-Hartl Contact Manajit Hayer-Hartl Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (430K) Supplementary Figures 1–3 and Supplementary Table 1 Additional data Entities in this article * Protein grpE grpE Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Ribulose bisphosphate carboxylase large chain rbcL Synechococcus sp. (strain ATCC 27144 / PCC 6301 / SAUG 1402/1) * View in UniProt * View in Entrez Gene * RbcX protein, rubisco chaperone rbcX Synechococcus sp. (strain ATCC 27264 / PCC 7002 / PR-6) * View in UniProt * View in Entrez Gene * RbcX protein, rubisco chaperone rbcX Synechococcus sp. (strain ATCC 27144 / PCC 6301 / SAUG 1402/1) * View in UniProt * View in Entrez Gene * Chaperone protein DnaJ dnaJ Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Chaperone protein DnaK dnaK Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * 60 kDa chaperonin groL Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Ribulose bisphosphate carboxylase small chain rbcS Synechococcus sp. (strain ATCC 27144 / PCC 6301 / SAUG 1402/1) * View in UniProt * View in Entrez Gene * RbcX protein, rubisco chaperone Anabaena sp. * View in UniProt * 10 kDa chaperonin groS Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene - Selective removal of promoter nucleosomes by the RSC chromatin-remodeling complex
- Nat Struct Mol Biol 18(8):881-885 (2011)
Nature Structural & Molecular Biology | Article Selective removal of promoter nucleosomes by the RSC chromatin-remodeling complex * Yahli Lorch1 * Joachim Griesenbeck1, 2 * Hinrich Boeger1, 2 * Barbara Maier-Davis1 * Roger D Kornberg1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:881–885Year published:(2011)DOI:doi:10.1038/nsmb.2072Received02 September 2010Accepted22 April 2011Published online03 July 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 Purified chromatin rings, excised from the PHO5 locus of Saccharomyces cerevisiae in transcriptionally repressed and activated states, were remodeled with RSC and ATP. Nucleosomes were translocated, and those originating on the promoter of repressed rings were removed, whereas those originating on the open reading frame (ORF) were retained. Treatment of the repressed rings with histone deacetylase diminished the removal of promoter nucleosomes. These findings point to a principle of promoter chromatin remodeling for transcription, namely that promoter specificity resides primarily in the nucleosomes rather than in the remodeling complex that acts upon them. View full text Figures at a glance * Figure 1: Effect of RSC and ATP on the accessibility of gene rings to digestion by BstEII and ClaI. ATP was destroyed with hexokinase (hexo) and glucose (glu) after RSC action and before restriction endonuclease digestion, as shown in lanes 3, 6, 9 and 12. Southern blot hybridization was done with probes described in Online Methods and diagrammed in Supplementary Figure 1. An arrow indicates the band due to uncut DNA. * Figure 2: Effect of RSC and ATP on the accessibility of PHO5 rings to restriction endonuclease digestion. () Gene rings. The slash (/) in "N-1/N-2" means BstEII cuts both N-1 and N-2; 3xlexA is three lexA binding sites, as detailed in references 9 and 24. () Promoter rings. The percentages of repressed (r) and activated (a) rings digested are tabulated and the percentage values for repressed rings are also shown in a bar graph. Black bars, no RSC treatment; light gray bars, treatment with RSC and ATP; dark gray bars, treatment with RSC and ATP, followed by destruction of ATP with hexokinase (hexo) and glucose (glu). nd, not determined. * Figure 3: Effect of RSC and ATP on topoisomer distributions of PHO5 rings. () Gene rings. () Promoter rings. RSC was at the level of 200 ng per reaction. Comp, competitor; hexo, hexokinase. * Figure 4: Nucleosome loss determined by limit nuclease digestion. Examples of limit micrococcal nuclease digestion experiments, conducted as described in Online Methods, with calculations as described in Results . Author information * Abstract * Author information * Supplementary information Affiliations * Department of Structural Biology, Stanford University School of Medicine, Stanford, California, USA. * Yahli Lorch, * Joachim Griesenbeck, * Hinrich Boeger, * Barbara Maier-Davis & * Roger D Kornberg * Present addresses: Universitaet Regensburg, Institut für Biochemie, Genetik und Mikrobiologie, Lehrstuhl Biochemie III, Regensburg, Germany (J.G.); Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, USA (H.B.). * Joachim Griesenbeck & * Hinrich Boeger Contributions Y.L. designed, conducted and interpreted experiments and wrote the paper. J.G. and H.B. participated in the isolation of chromatin rings and in limit nuclease digestion experiments. B.M.-D. isolated chromatin rings and RSC. R.D.K. designed and interpreted experiments and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Yahli Lorch Author Details * Yahli Lorch Contact Yahli Lorch Search for this author in: * NPG journals * PubMed * Google Scholar * Joachim Griesenbeck Search for this author in: * NPG journals * PubMed * Google Scholar * Hinrich Boeger Search for this author in: * NPG journals * PubMed * Google Scholar * Barbara Maier-Davis Search for this author in: * NPG journals * PubMed * Google Scholar * Roger D Kornberg Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2.6M) Supplementary Figures 1–5 Additional data Entities in this article * Repressible acid phosphatase PHO5 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Galactokinase GAL1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Histone H2A.Z H2AFZ Homo sapiens * View in UniProt * View in Entrez Gene * Histone deacetylase 6 HDAC6 Homo sapiens * View in UniProt * View in Entrez Gene * Bifunctional protein GAL10 GAL10 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Nucleosome assembly protein NAP1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Phosphate system positive regulatory protein PHO4 PHO4 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Histone H2A.Z HTZ1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Chromatin structure-remodeling complex protein RSC3 RSC3 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene - Modular mechanism of Wnt signaling inhibition by Wnt inhibitory factor 1
- Nat Struct Mol Biol 18(8):886-893 (2011)
Nature Structural & Molecular Biology | Article Modular mechanism of Wnt signaling inhibition by Wnt inhibitory factor 1 * Tomas Malinauskas1 * A Radu Aricescu1 * Weixian Lu1 * Christian Siebold1 * E Yvonne Jones1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:886–893Year published:(2011)DOI:doi:10.1038/nsmb.2081Received19 October 2010Accepted06 May 2011Published online10 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 Wnt morphogens control embryonic development and homeostasis in adult tissues. In vertebrates the N-terminal WIF domain (WIF-1WD) of Wnt inhibitory factor 1 (WIF-1) binds Wnt ligands. Our crystal structure of WIF-1WD reveals a previously unidentified binding site for phospholipid; two acyl chains extend deep into the domain, and the head group is exposed to the surface. Biophysical and cellular assays indicate that there is a WIF-1WD Wnt-binding surface proximal to the lipid head group but also implicate the five epidermal growth factor (EGF)-like domains (EGFs I–V) in Wnt binding. The six-domain WIF-1 crystal structure shows that EGFs I–V are wrapped back, interfacing with WIF-1WD at EGF III. EGFs II–V contain a heparan sulfate proteoglycan (HSPG)-binding site, consistent with conserved positively charged residues on EGF IV. This combination of HSPG- and Wnt-binding properties suggests a modular model for the localization of WIF-1 and for signal inhibition within morp! hogen gradients. View full text Figures at a glance * Figure 1: The WIF-1WD-EGF-I structure and lipid-binding cavity. () Schematic domain organization of human WIF-1. Glycosylation sites linked to Asn88, Asn245 and Thr255 are marked by hexagons. The WIF domain is colored in green-to-blue transition, and EGFs I–III are colored magenta, yellow and red, respectively. The amino acid sequence boundaries of the WIF-1 constructs are shown below. () Ribbon diagram of WIF-1WD-EGF-I in two views that differ by a 90° rotation about a vertical axis. WIF-1WD–EGF-I is colored as in . Asn88-linked N-acetylglucosamine (GlcNAc, gray) and disulfide bridges (orange) are shown as sticks. DPPC is shown as spheres (C, gray; N, blue; O, red; P, orange). () The phosphatidylcholine head group of DPPC (shown as sticks) exposed to the solvent with color coding as in (except C in light blue). The composite omit electron density map (contoured at 1σ) is shown as gray mesh. () The WIF domain lipid-binding pocket (gray surface; DPPC shown as sticks with color coding as in ). Residues within 4.5 Å of DPPC are shown! as gray sticks. () MALDI-TOF mass spectrum of WIF-1–bound ligands. The length and saturation of the lipid acyl chains are shown in parentheses. * Figure 2: The contributions of the WIF and EGF-like domains to Wnt inhibition. () The concentration-dependent inhibition of Wnt3a signaling in a cellular assay by four constructs of WIF-1: WIF-1ΔC (black), WIF-1WD–EGF-I (blue), WIF-1WD (green) and WIF-1EGFs I–V (magenta). The concentration of human Wnt3a was kept constant at 4.7 nM. The dashed line indicates endogenous Wnt signaling in HEK293T cells without added Wnt3a. Experiments were performed in triplicate, and error bars show s.d. () Binding of WIF-1 constructs and BSA (control) to mouse Wnt3a. Shown are WIF-1Full length (); WIF-1WD–EGF-I (); WIF-1WD (); WIF-1EGFs I–V (); and BSA (). Different concentrations of WIF-1 constructs were injected over surface coupled with mouse Wnt3a. Insets, representative plots of the binding response (response units (RU)) derived from the sensorgrams as a function of WIF-1 construct (or BSA) concentration. Kd values (±s.d.) calculated from three independent experiments are shown. * Figure 3: A discontinuous Wnt-binding site on the WIF domain. () Inhibition of Wnt3a signaling by mutation-bearing (left) and wild-type WIF-1 constructs (right). Inhibition of each WIF-1 construct was compared to the wild-type WIF-1ΔC in the cellular assay. Higher ratio (0.81) of Wnt signal (wild-type WIF-1ΔC/WIF-1 variant) corresponds to strong inhibition and lower (0.20.5) to poor inhibition. Inhibition of Wnt3a (4.7 nM) signaling was tested in the presence of 33 nM (gray bar) and 500 nM (black bar) of the WIF-1 variant. Yellow hexagons indicate mutations predicted to introduce N-linked glycosylation sites. () Residues tested in the cellular assay for Wnt3a signaling inhibition mapped on the surface of the WIF domain. DPPC (blue) and GlcNAc (yellow) are shown as balls and sticks. Residues for which mutations resulted in poor, intermediate and strong inhibition of Wnt3a signaling are colored red, orange and purple, respectively; Phe174 and Ile172 are colored red and orange, respectively. A hydrophobic patch on the surface of the WIF! domain (180° view) is marked with an asterisk. () The surface of the WIF domain colored by residue conservation (conserved, magenta; variable, cyan). Fourteen Wnt-binding WIF domains (Supplementary Fig. 3b) are included in the sequence conservation analysis. WIF domains from Hh-binding Shifted are excluded. A potential Wnt3a-binding surface is circled. () WIF-1WD–EGF-I wild-type (left) and WIF-1WD–EGF-I Met77Trp (middle) crystal structures are superimposed on each other (right). DPPC is shown as sticks. * Figure 4: The structure of WIF-1ΔC. () Ribbon diagram of WIF-1ΔC. The orientation of the WIF domain is the same as in Figure 1b. The WIF domain and EGFs I–III are colored light blue, magenta, yellow and red, respectively. Ten disulfide bridges are shown as gray sticks and marked with Roman numerals. DPPC is shown as spheres. () Electrostatic properties of WIF-1ΔC. The protein is shown as solvent-accessible surface colored by electrostatic potential contoured at ± 8 kT/e (red, acidic; blue, basic). GlcNAc moieties on Asn88 (WIF domain) and Asn245 (EGF III) are shown as yellow spheres. A potential Wnt3a-binding surface is circled. () Orthogonal relative orientation of the DPPC head group between superimposed structures of WIF-1ΔC (light blue; DPPC colored as in Fig. 1c,d) and WIF-1WD–EGF-I (green). () Hydrophobic contact formed between the WIF domain hydrophobic patch (marked with an asterisk in Fig. 3b, 180° view) and EGF III. Interacting residues are shown as sticks. * Figure 5: HSPG binding contributes to a modular mechanism for WIF-1 function. () Homology model of the human WIF-1 EGF IV as a ribbon diagram. Disulfide bridges XIXIII are shown as orange sticks. () Electrostatic properties of WIF-1 EGF IV model. The protein is shown as solvent-accessible surface colored by electrostatic potential contoured at ±8 kT/e (red, acidic; blue, basic). (,) Chemical structures of heparin () and heparan sulfate (; left), binding sensorgrams of WIF-1ΔC to heparin and heparan sulfate (middle), and representative plots of the binding response (response units (RU)) derived from the sensorgram as a function of WIF-1ΔC concentration (right). Kd values (± s.d.) calculated from three independent experiments are shown. () The Wnt–β-catenin pathway is initiated at the cell surface when secreted Wnt3a (black star) forms a ternary complex with the cell-surface receptors Frizzled (blue) and LRP5/6 (red). This results in downstream signal transduction and activation of Wnt target genes. HSPGs (gray) on the cell surface bind to Wnts a! nd modulate their extracellular gradient. () The combination of HSPG- and Wnt-binding properties provides a modular mechanism for controlling the localization of WIF-1 (green) and hence Wnt-signal inhibition within the Wnt concentration gradient. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 2YGQ * 2YGO * 2YGP * 2YGN * 2YGQ * 2YGO * 2YGP * 2YGN Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK. * Tomas Malinauskas, * A Radu Aricescu, * Weixian Lu, * Christian Siebold & * E Yvonne Jones Contributions T.M., A.R.A., C.S. and E.Y.J. designed the project. T.M. performed all the experiments. W.L. contributed to WIF-1 protein expression. A.R.A. and C.S. contributed to X-ray data collection and analysis. T.M., A.R.A., C.S. and E.Y.J. analyzed the data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Christian Siebold or * E Yvonne Jones Author Details * Tomas Malinauskas Search for this author in: * NPG journals * PubMed * Google Scholar * A Radu Aricescu Search for this author in: * NPG journals * PubMed * Google Scholar * Weixian Lu Search for this author in: * NPG journals * PubMed * Google Scholar * Christian Siebold Contact Christian Siebold Search for this author in: * NPG journals * PubMed * Google Scholar * E Yvonne Jones Contact E Yvonne Jones 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 (2M) Supplementary Methods and Supplementary Figures 1–9 Additional data Entities in this article * Protein Wnt-4 WNT4 Homo sapiens * View in UniProt * View in Entrez Gene * Protein Wnt-3a WNT3A Homo sapiens * View in UniProt * View in Entrez Gene * Protein Wnt-7a WNT7A Homo sapiens * View in UniProt * View in Entrez Gene * Protein Wnt-5a WNT5A Homo sapiens * View in UniProt * View in Entrez Gene * Low-density lipoprotein receptor-related protein 5 LRP5 Homo sapiens * View in UniProt * View in Entrez Gene * Catenin beta-1 CTNNB1 Homo sapiens * View in UniProt * View in Entrez Gene * Cerberus CER1 Homo sapiens * View in UniProt * View in Entrez Gene * Low-density lipoprotein receptor-related protein 6 LRP6 Homo sapiens * View in UniProt * View in Entrez Gene * Protein hedgehog hh Drosophila melanogaster * View in UniProt * View in Entrez Gene * Protein wingless wg Drosophila melanogaster * View in UniProt * View in Entrez Gene * Protein shifted shf Drosophila melanogaster * View in UniProt * View in Entrez Gene * Protein Wnt-11 WNT11 Homo sapiens * View in UniProt * View in Entrez Gene * Dickkopf-related protein 1 DKK1 Homo sapiens * View in UniProt * View in Entrez Gene * Protein Wnt-9a WNT9A Homo sapiens * View in UniProt * View in Entrez Gene * Sclerostin SOST Homo sapiens * View in UniProt * View in Entrez Gene * Wnt inhibitory factor 1 WIF1 Homo sapiens * View in UniProt * View in Entrez Gene * Wnt inhibitory factor 1 Wif1 Mus musculus * View in UniProt * View in Entrez Gene * Secreted frizzled-related protein 1 SFRP1 Homo sapiens * View in UniProt * View in Entrez Gene * Wnt inhibitory factor 1 wif1 Xenopus laevis * View in UniProt * View in Entrez Gene * Noelin OLFM1 Homo sapiens * View in UniProt * View in Entrez Gene * Protein Wnt-8 wnt8a Xenopus laevis * View in UniProt * View in Entrez Gene * Abnormal cell lineage protein 18, confirmed by transcript evidence lin-18 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Wnt inhibitory factor 1 wif1 Danio rerio * View in UniProt * View in Entrez Gene * Protein Wnt-3a Wnt3a Mus musculus * View in UniProt * View in Entrez Gene - Condensin structures chromosomal DNA through topological links
- Nat Struct Mol Biol 18(8):894-901 (2011)
Nature Structural & Molecular Biology | Article Condensin structures chromosomal DNA through topological links * Sara Cuylen1 * Jutta Metz1 * Christian H Haering1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:894–901Year published:(2011)DOI:doi:10.1038/nsmb.2087Received15 December 2010Accepted10 May 2011Published online17 July 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 The multisubunit condensin complex is essential for the structural organization of eukaryotic chromosomes during their segregation by the mitotic spindle, but the mechanistic basis for its function is not understood. To address how condensin binds to and structures chromosomes, we have isolated from Saccharomyces cerevisiae cells circular minichromosomes linked to condensin. We find that either linearization of minichromosome DNA or proteolytic opening of the ring-like structure formed through the connection of the two ATPase heads of condensin's structural maintenance of chromosomes (SMC) heterodimer by its kleisin subunit eliminates their association. This suggests that condensin rings encircle chromosomal DNA. We further show that release of condensin from chromosomes by ring opening in dividing cells compromises the partitioning of chromosome regions distal to centromeres. Condensin hence forms topological links within chromatid arms that provide the arms with the struct! ural rigidity necessary for their segregation. View full text Figures at a glance * Figure 1: Linearization releases minichromosome DNA from condensin. () Co-immunoprecipitation of 4.3 kb minichromosomes from lysates of asynchronous yeast cultures (strains C2292, C2293, C2350) with condensin (Brn1-HA6) or cohesin (Scc1-HA6) was tested by Southern blotting of input (IN), flow-through (FT) and immunoprecipitated fractions (B, concentrated 5× or 25× relative to input). Relaxed (oval) or supercoiled (braided oval) monomers and supercoiled concatemers (*) are indicated (see Supplementary Fig. 1c). 1.6% or 0.7% of total minichromosome DNA was co-immunoprecipitated with condensin or cohesin, respectively (compare to 0.02% when neither was tagged). (,) Linearization of minichromosome DNAs should cause them to slide out of condensin rings if they were topologically bound but should not affect binding if they were bound through direct protein-chromatin contacts. () Linear minichromosome DNA (squiggle) released from bead-immobilized condensin (top, strain C2292) or cohesin (middle, strain C2350) after BglII cleavage into supernatant! (SUP) or wash fractions of increasing salt concentrations (0–1 M NaCl), or still bound to beads (SB) was detected by Southern blotting and quantified (bottom, mean ± s.d. of 3 experiments). () As in after cleavage at a DraIII site opposite to the BglII site. * Figure 2: Ring opening by TEV cleavage of Brn1 eliminates condensin function. () TEV sites were introduced between the N-terminal Smc2 and C-terminal Smc4 binding motifs (HTH or WHD) into Brn1 at positions of low secondary structure probability. () TEV protease expression was induced from the GAL1 promoter (pGAL1) in asynchronous cultures (strains C2437, C2320, C2439, C2322, C2455 and C2335) and Brn1 cleavage was monitored by western blotting against C-terminal HA6 tags in whole cell extracts at the indicated times after induction. () Brn1(TEV) constructs complement brn1Δ in the absence of TEV expression but—with the exception of Brn1(TEV251)—not after induction of TEV expression (strains as in , plus C2324 and K8758). () TEV251 is positioned in the center of the binding site for Ycs4 in Brn1. It is therefore possible that Ycs4 connects the Brn1(TEV251) cleavage fragments to keep condensin rings intact, whereas cleavage at other TEV sites opens the ring. () In vivo TEV cleavage of Brn1(TEV251) and Ycs4(TEV829) after TEV protease induction was mon! itored by western blotting against C-terminal HA6 or PK9 tags, respectively (strains C2813, C2820, C2805). () Simultaneous cleavage of Brn1(TEV251) and Ycs4(TEV829), but not cleavage of either protein alone, destroys condensin function at 37 °C. * Figure 3: Ring opening by Brn1 cleavage releases condensin from minichromosomes. () If minichromosome DNAs were encircled within condensin rings, they should be released when rings are opened after cleavage of Brn1(TEV622) but not when ring integrity remains intact after cleavage of Brn1(TEV251). () Immobilized condensin– and cohesin–minichromosome complexes (immunoprecipitated from strains C2461, C2460, C2348 and C2349) were incubated with recombinant TEV protease. Cleavage was monitored by western blotting against the C-terminal HA6 tag on Brn1 or Scc1. Small circle indicates Scc1separase cleavage fragment. () Release of closed circular minichromosome DNA after TEV cleavage was detected by Southern blotting of supernatant (SUP), salt wash (0–1 M NaCl) or still-bound (SB) fractions and quantified (mean ± s.d.) of 3 experiments. Symbols as in Figure 1. * Figure 4: Ring opening by Brn1 cleavage in vivo releases condensin from chromosomes. () Completion of Brn1 or Scc1 cleavage 4 h after TEV induction in asynchronous cultures (strains C2463, C2567, C2443 and C2444) was confirmed by western blotting and co-immunoprecipitation of 7.9-kb minichromosomes with HA-tagged condensin or cohesin, respectively, and was assayed by Southern blotting of input (IN), supernatant (SUP) and bound fractions (B). Symbols as in Figure 1. () Cleavage of Brn1 3 h after TEV induction in cells arrested with nocodazole (strains C2783, C2781 and C2864) was tested by western blotting as in , and condensin release from chromosomes was assayed by immunofluorescence staining of chromosome spreads (red, anti-HA; blue, 4′,6-diamidino-2-phenylindole (DAPI); scale bar, 5 μm). Chromosomal Brn1-HA6 immunofluorescence signals were quantified; horizontal lines define the median, boxes define the 25th and 75th percentiles, and whiskers define the 10th and 90th percentiles. () TEV protease expression was induced in asynchronous cultures (strains C! 2439, C2455, C2783, C2781 and K9872) and Brn1 cleavage monitored 3 h after induction as in . Levels of Brn1-HA6 or Smc2-PK6 bound to two chromosomal condensin sites (left, 5′ UTR of RDN37 rDNA; right, CEN4 centromere) before and after Brn1 cleavage were measured by anti-HA and anti-PK chromatin immunoprecipitation (ChIP) followed by quantitative PCR (mean ± s.d. of at least 3 experiments). * Figure 5: Ring opening by cleavage of Smc4 in vivo releases condensin from chromosomes. () Opening of condensin rings by TEV cleavage of both strands of Smc4's coiled coil at juxtaposed positions. () Smc4 cleavage was monitored by western blotting of whole cell extracts 4 h after TEV protease induction in asynchronous cultures (strains C2838, C2857, C2859 and C2864) against C-terminal PK6 tags in whole cell extracts. Blotting against the Flag epitope preceding the TEV sites confirmed that Smc4(TEV552/971) had been cleaved at both sites. () Smc4 coiled-coil cleavage (but not merely 'nicking') after induction of TEV expression destroys condensin function (strains as in ). () TEV protease expression was induced in asynchronous cultures (strains C2838, C2859, C2857 and K9872) grown at 25 °C, and Smc4 cleavage was monitored 4 h after induction by western blotting against PK6 and Flag tags. Levels of Smc4 bound to the 5′ UTR of RDN37 rDNA (top) and CEN4 (bottom) before and after Smc4 cleavage were measured by anti-PK ChIP followed by qPCR (mean ± s.d. of at least! 2 experiments). * Figure 6: Condensin release by ring opening prevents chromosome arm segregation. (–) Brn1 cleavage during G1 phase. TEV protease expression from pGAL1 was induced in cells arrested in G1 phase by α-factor. Cells were released from the arrest 3 h after protease induction, and chromosome segregation was recorded by live-cell imaging. Cell cycle progression was measured by FACScan analysis of DNA content, and Brn1 cleavage during the α-factor arrest was monitored by western blotting. Segregation of GFP-labeled repeats ~20 kb (strains C2381 and C2665) or ~400 kb (strains C2481 and C2619) from centromere V or of the rDNA marked by Net1-GFP (strains C2497 and C2621), was scored according to the categories shown. (–) Brn1 cleavage during metaphase. TEV protease expression was induced in cells arrested in metaphase by repression of Cdc20 expression from the MET3 promoter (pMET3). Cells were released into methionine-free medium 3 h after protease induction, and chromosome segregation was recorded by live-cell imaging as in (strains C2628, C2666, C2513, C261! 8, C2484 and C2620). * Figure 7: Structuring of chromosomes through topological condensin links. () Condensin complexes may structurally reinforce chromosome arms into rigid bodies that can be moved by mitotic spindle microtubules connected to a single kinetochore. Loss of rigidity triggered by release of condensin would cause chromosome arms to get stretched and lag behind centromeres during segregation. () Condensin rings may link different chromosome segments by encircling one chromatid segment while binding directly to a second segment of the same chromatid. The topologically bound segment may be free to slide through the ring. () Alternatively, condensin rings may encircle both segments within their ring structure. Author information * Abstract * Author information * Supplementary information Affiliations * European Molecular Biology Laboratory (EMBL), Cell Biology & Biophysics Unit, Heidelberg, Germany. * Sara Cuylen, * Jutta Metz & * Christian H Haering Contributions S.C., J.M. and C.H.H., yeast strain and plasmid generation; S.C., minichromosome, co-immunoprecipitation and ChIP-qPCR experiments; S.C. and J.M., live-cell imaging experiments; C.H.H., chromosome spreads; S.C. and C.H.H., project design and manuscript preparation; C.H.H., project supervision. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Christian H Haering Author Details * Sara Cuylen Search for this author in: * NPG journals * PubMed * Google Scholar * Jutta Metz Search for this author in: * NPG journals * PubMed * Google Scholar * Christian H Haering Contact Christian H Haering Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (5M) Supplementary Figures 1–5, Supplementary Tables 1 and 2, and Supplementary Methods Additional data Entities in this article * Condensin complex subunit 3 YCG1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Condensin complex subunit 1 YCS4 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Condensin-2 complex subunit D3 NCAPD3 Homo sapiens * View in UniProt * View in Entrez Gene * Condensin complex subunit 1 NCAPD2 Homo sapiens * View in UniProt * View in Entrez Gene * Condensin complex subunit 2 BRN1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Structural maintenance of chromosomes protein 4 SMC4 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Condensin-2 complex subunit H2 NCAPH2 Homo sapiens * View in UniProt * View in Entrez Gene * Condensin complex subunit 2 NCAPH Homo sapiens * View in UniProt * View in Entrez Gene * Structural maintenance of chromosomes protein 1 SMC1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Condensin-2 complex subunit G2 NCAPG2 Homo sapiens * View in UniProt * View in Entrez Gene * Condensin complex subunit 3 NCAPG Homo sapiens * View in UniProt * View in Entrez Gene * Structural maintenance of chromosomes protein 2 SMC2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * APC/C activator protein CDC20 CDC20 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Galactokinase GAL1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Sister chromatid cohesion protein 1 MCD1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Structural maintenance of chromosomes protein 3 SMC3 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Separin ESP1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Sulfate adenylyltransferase MET3 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Nucleolar protein NET1 NET1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene - Pinkbar is an epithelial-specific BAR domain protein that generates planar membrane structures
- Nat Struct Mol Biol 18(8):902-907 (2011)
Nature Structural & Molecular Biology | Article Pinkbar is an epithelial-specific BAR domain protein that generates planar membrane structures * Anette Pykäläinen1 * Malgorzata Boczkowska2 * Hongxia Zhao1 * Juha Saarikangas1 * Grzegorz Rebowski2 * Maurice Jansen3 * Janne Hakanen4 * Essi V Koskela1 * Johan Peränen1 * Helena Vihinen1 * Eija Jokitalo1 * Marjo Salminen4 * Elina Ikonen3 * Roberto Dominguez2 * Pekka Lappalainen1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:902–907Year published:(2011)DOI:doi:10.1038/nsmb.2079Received12 October 2010Accepted05 May 2011Published online10 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 Bin/amphipysin/Rvs (BAR)-domain proteins sculpt cellular membranes and have key roles in processes such as endocytosis, cell motility and morphogenesis. BAR domains are divided into three subfamilies: BAR– and F-BAR–domain proteins generate positive membrane curvature and stabilize cellular invaginations, whereas I-BAR–domain proteins induce negative curvature and stabilize protrusions. We show that a previously uncharacterized member of the I-BAR subfamily, Pinkbar, is specifically expressed in intestinal epithelial cells, where it localizes to Rab13-positive vesicles and to the plasma membrane at intercellular junctions. Notably, the BAR domain of Pinkbar does not induce membrane tubulation but promotes the formation of planar membrane sheets. Structural and mutagenesis analyses reveal that the BAR domain of Pinkbar has a relatively flat lipid-binding interface and that it assembles into sheet-like oligomers in crystals and in solution, which may explain its unique m! embrane-deforming activity. View full text Figures at a glance * Figure 1: Endogenous Pinkbar associates with membranes in Caco-2 cells and colocalizes with the small GTPase Rab13. (,) The association of endogenous Pinkbar (molecular weight 58.4 kDa) with cellular membranes was studied by sucrose gradient centrifugation of Caco-2 cell lysate. Fractions (1 ml) were collected from top (fraction 1) to bottom (fraction 9). Pinkbar is present at the 10–65% interface of the sucrose gradient in the same fractions (4 and 5) as the membranes and the positive control, E-cadherin (molecular weight 97.5 kDa). Protein is indicated by arrows. (,) Immuno-EM sections showing the localization of endogenous Pinkbar in Caco-2 cells (day 16). Pinkbar (indicated by arrows) localizes to the plasma membrane at cell-cell junctions () and to intracellular vesicle clusters (). Scale bars show 200 nm () and 100 nm (). () In fully polarized Caco-2 cells (day 16) endogenous Pinkbar show partial colocalization with endogenous Rab13 in punctuate cytoplasmic structures, as detected by immunofluorescence microscopy. Insets display the localization of Pinkbar and Rab13 at higher magn! ification. Scale bar, 10 μm. * Figure 2: The BAR domain of Pinkbar promotes the formation of planar membrane structures. () Three-dimensional electron tomography analysis of a membrane sheet network induced by the BAR domain of Pinkbar. Left, tomographic slice from reconstruction of a section 250 nm thick, with a magnification of the selected region shown as an inset. Scale bars, 400 nm and 50 nm, respectively. Arrowheads indicate highly curved membrane areas, which lack higher electron density attributable to Pinkbar. Right, a three-dimensional model obtained from the same structure in which few of the sheets that were not connected with other membranes in the reconstructed volume were removed. () Giant unilamellar vesicles (GUVs) containing fluorescent nitrobenzoxadiazole (NBD)-labeled phosphatidylcholine (PC) (green) were incubated with mCherry-labeled BAR domain of Pinkbar (red). In the absence of protein, the vesicles were predominantly spherical; in contrast, BAR domain–decorated vesicles often showed abnormal morphology and flat surfaces. The lipid composition of the GUVs used in the ! assay was POPC:POPE:POPS:PIP2:NBD-PC (49:20:20:10:1) and the protein concentration was 100 nM. Scale bars, 10 μm. () Easy3D (Imaris 7.0.0, Bitplane) illustration of flat indentations on the surface of a GUV produced by the BAR domain of Pinkbar. BAR-mCherry is shown in red and NBD-PC in green. Scale bar, 10 μm. () Quantification of the proportion of deformed vesicles in GUVs decorated with the BAR domain of Pinkbar, GUVs from the same experiment that did not accumulate Pinkbar and GUVs from samples treated with buffer only. The data are from three independent experiments; error bars represent s.d. w/o, without. * Figure 3: Crystal structure and oligomerization of the BAR domain of Pinkbar. () Ribbon diagram representation of the structure of the BAR domain of Pinkbar. The two chains that form the BAR domain are shown in yellow and blue. Each chain contains three α-helices, which are kinked toward their middle (see also Supplementary Fig. 6) and are thus labeled as α1a-α1b to α3a-α3b (or α1a′-α1b′ to α3a′-α3b′ for the second chain). () Electrostatic surface representation of the BAR domain of Pinkbar compared to those of IRSp53 and MIM. Blue and red indicate negatively and positively charged regions, respectively. Note that the BAR domain of Pinkbar is shorter and its membrane-binding surface (highlighted by a gray background) less curved than those of IRSp53 and MIM. () The BAR domain of Pinkbar, which forms sheet-like membrane structures in solution, also forms a planar oligomer in the crystal lattice, in which the membrane-binding interface of individual BAR domains all face in the same direction (that is, facing the reader in the view shown! on the left). The green background highlights the boundaries of a single BAR domain dimer. This planar oligomer is somewhat reminiscent of the planar BAR-BAR coats observed by EM tomography of F-BAR domains13. () The conspicuously exposed residue Trp141 may be involved in the stabilization of the lateral oligomer (the ribbon diagram is shown in a similar orientation as in , left), as suggested by light-scattering analysis in solution of wild-type and W141S mutant BAR domain constructs (Supplementary Fig. 7a,b). * Figure 4: Mechanism of membrane binding and deformation by the BAR domain of Pinkbar. () Effect of mutations of the BAR domain of Pinkbar on the clustering of BODIPY-TMR-PI(4,5)P2 measured by fluorometric assay. Double or triple mutations of the positively charged residues (K109A K116A, R127A K135A and R145A K146A R147A) had the strongest effect on membrane binding (red). A moderate effect was observed for mutant I124S (orange). The triple mutant K149 R152 K155 and the single mutants W141S and L214S had no significant effect on PI(4,5)P2 clustering (green). The composition of the liposomes used in the assay was POPC:POPE:POPS:PIP2:BODIPY-PIP2 (50:20:20:9.5:0.5) and the concentration of protein was 1.6 μM. Each bar represents the mean of three individual measurements; error bars represent s.d. () The residues mutated in this study are shown as ball and stick in a ribbon diagram of the structure of the BAR domain of Pinkbar and colored as in . Note that the residues that had the strongest effects on BODIPY quenching cluster along the membrane-binding surface o! f the BAR domain. () The membrane-binding surfaces of individual BAR domains all face in the same direction in the planar oligomer of the crystal lattice and thus the lipid-binding interface in the oligomer is relatively flat. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3OK8 * 3OK8 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Institute of Biotechnology, University of Helsinki, Helsinki, Finland. * Anette Pykäläinen, * Hongxia Zhao, * Juha Saarikangas, * Essi V Koskela, * Johan Peränen, * Helena Vihinen, * Eija Jokitalo & * Pekka Lappalainen * Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * Malgorzata Boczkowska, * Grzegorz Rebowski & * Roberto Dominguez * Institute of Biomedicine, University of Helsinki, Helsinki, Finland. * Maurice Jansen & * Elina Ikonen * Faculty of Veterinary Medicine, Department of Veterinary Medicine, Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland. * Janne Hakanen & * Marjo Salminen Contributions A.P., M.B., H.Z., J.S., G.R., M.J., J.H., E.V.K., H.V. and R.D. performed the experiments. A.P., J.P., E.J., M.S., E.I., R.D. and P.L designed and supervised the project. A.P., R.D. and P.L. prepared the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Pekka Lappalainen or * Roberto Dominguez Author Details * Anette Pykäläinen Search for this author in: * NPG journals * PubMed * Google Scholar * Malgorzata Boczkowska Search for this author in: * NPG journals * PubMed * Google Scholar * Hongxia Zhao Search for this author in: * NPG journals * PubMed * Google Scholar * Juha Saarikangas Search for this author in: * NPG journals * PubMed * Google Scholar * Grzegorz Rebowski Search for this author in: * NPG journals * PubMed * Google Scholar * Maurice Jansen Search for this author in: * NPG journals * PubMed * Google Scholar * Janne Hakanen Search for this author in: * NPG journals * PubMed * Google Scholar * Essi V Koskela Search for this author in: * NPG journals * PubMed * Google Scholar * Johan Peränen Search for this author in: * NPG journals * PubMed * Google Scholar * Helena Vihinen Search for this author in: * NPG journals * PubMed * Google Scholar * Eija Jokitalo Search for this author in: * NPG journals * PubMed * Google Scholar * Marjo Salminen Search for this author in: * NPG journals * PubMed * Google Scholar * Elina Ikonen 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 * Pekka Lappalainen Contact Pekka Lappalainen 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 (2M) Supplementary Figures 1–9 and Supplementary Methods Additional data Entities in this article * Brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 2 Baiap2l2 Mus musculus * View in UniProt * View in Entrez Gene * Brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 1 BAIAP2L1 Homo sapiens * View in UniProt * View in Entrez Gene * MTSS1-like protein MTSS1L Homo sapiens * View in UniProt * View in Entrez Gene * Cadherin-1 CDH1 Homo sapiens * View in UniProt * View in Entrez Gene * SLIT-ROBO Rho GTPase-activating protein 2 SRGAP2 Homo sapiens * View in UniProt * View in Entrez Gene * Ras-related protein Rab-13 RAB13 Homo sapiens * View in UniProt * View in Entrez Gene * Brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 2 BAIAP2L2 Homo sapiens * View in UniProt * View in Entrez Gene * Brain-specific angiogenesis inhibitor 1-associated protein 2 BAIAP2 Homo sapiens * View in UniProt * View in Entrez Gene * Metastasis suppressor protein 1 MTSS1 Homo sapiens * View in UniProt * View in Entrez Gene - Dimerization of Plasmodium vivaxDBP is induced upon receptor binding and drives recognition of DARC
- Nat Struct Mol Biol 18(8):908-914 (2011)
Nature Structural & Molecular Biology | Article Dimerization of Plasmodium vivaxDBP is induced upon receptor binding and drives recognition of DARC * Joseph D Batchelor1 * Jacob A Zahm1 * Niraj H Tolia1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:908–914Year published:(2011)DOI:doi:10.1038/nsmb.2088Received03 February 2011Accepted11 May 2011Published online10 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 Plasmodium vivax and Plasmodium knowlesi invasion depends on the parasite Duffy-binding protein DBL domain (RII-PvDBP or RII-PkDBP) engaging the Duffy antigen receptor for chemokines (DARC) on red blood cells. Inhibition of this key interaction provides an excellent opportunity for parasite control. There are competing models for whether Plasmodium ligands engage receptors as monomers or dimers, a question whose resolution has profound implications for parasite biology and control. We report crystallographic, solution and functional studies of RII-PvDBP showing that dimerization is required for and driven by receptor engagement. This work provides a unifying framework for prior studies and accounts for the action of naturally acquired blocking antibodies and the mechanism of immune evasion. We show that dimerization is conserved in DBL-domain receptor engagement and propose that receptor-mediated ligand dimerization drives receptor affinity and specificity. Because dimerizat! ion is prevalent in signaling, our studies raise the possibility that induced dimerization may activate pathways for invasion. View full text Figures at a glance * Figure 1: RII-PvDBP is composed of three subdomains, and a sulfotyrosine pocket within a DARC-binding groove is formed by the RII-PvDBP dimer. () RII-PvDBP separated into the three subdomains. Subdomain 1 (S1; red) contains the β-hairpin, subdomain 2 (S2; blue) is a four-helix bundle and subdomain 3 (S3; green) forms a second helical bundle. () The RII-PvDBP dimer in ribbon representation. Monomers are in green and yellow. The DARC-binding groove is outlined by a dashed box, and the dimer interface is indicated by a solid line. () Electrostatic mapping of the RII-PvDBP dimer. The DARC-binding groove is positively charged. () Density that clearly identifies selenate or phosphate at the RII-PvDBP dimer interface is shown. Left: selenium anomalous difference map (blue mesh) from crystals grown in the presence of selenate, contoured at 4σ. Middle: the difference map (green mesh), contoured at 2.5σ, of the crystals grown in phosphate before addition of phosphates. Right: the omit map (green mesh), contoured at 2.5σ, of the final refined structure with the phosphates omitted. () The putative sulfotyrosine-binding poc! ket and the Arg274-Glu249 salt bridge shown. For and , phosphates are drawn in stick form and colored red and yellow, side chains of residues involved in interactions are shown in stick form, and contacts are depicted by dashed black lines. () Percentage of cells expressing point mutants of RII-PvDBP that bind RBCs relative to wild type, shown with s.e.m. A paired two-tailed Student's t-test indicated that all mutants as compared to wild type have P < 0.0001. White bar, wild type; black bars, dimer mutants and rescue; gray bars, sulfotyrosine-binding mutants. * Figure 2: DARC binding drives dimerization of RII-PvDBP. Experimental (black) and theoretical SAXS plots of scattering intensity (I) against scattering momentum (Q) for the monomer (blue) and dimer (red) at different concentrations. An expanded plot of the low-angle data (0 < Q < 0.1) that clearly delineates oligomeric state is shown in the top right insert. Ab initio reconstructions are overlaid on structures (bottom left insert) with monomers colored in green and yellow and molecular envelopes in sand. () RII-PvDBP at 1 mg ml−1. () RII-PvDBP at 6 mg ml−1. () RII-PvDBP–DARC1–60 at 1 mg ml−1. () RII-PvDBP–DARC1–60 at 6 mg ml−1. * Figure 3: The sulfotyrosine pocket, DARC-binding groove and dimer interface are under selective pressure and are targeted by blocking antibodies. Monomers are in green and yellow. () Polymorphic residues20, 23 (blue) are excluded from the dimer interface but are evenly distributed over the remaining RII-PvDBP surface. () Amino acid substitutions that abrogate RBC rosetting25, 26 (purple) map to the DARC-binding groove and dimer interface. () Overlay of polymorphic residues (blue) and critical receptor-binding residues (purple) on the dimer. The DARC-binding groove at the dimer interface is composed of essential residues and devoid of polymorphisms. () Epitopes recognized by blocking antibodies22 (red, most significant; brown, significant) map to the functional regions of RII-PvDBP, which include the dimer interface and DARC-binding groove. () The minimal binding domain of RII-PvDBP (residues 256–426)27 contains the full dimer interface and DARC-binding groove. () A global view of the dimer, which shows that the asymmetric flap is disordered in chain A. Essential residues are colored in purple. () A detailed view of ! the asymmetric flap shows that this region contains several essential residues, suggesting a second potential DARC-binding site. * Figure 4: RII-PvDBP's dimer interface and receptor binding site are conserved in VAR2CSA DBL6ɛ. () Examination of the crystal packing interfaces for VAR2CSA DBL6ɛ revealed a dimeric organization identical to the RII-PvDBP dimer. Critical VAR2CSA DBL6ɛ binding residues are shown in red and map to the putative sulfotyrosine-binding pocket, indicating that both the receptor-binding pocket and dimer interface are conserved in these two DBL domains. Monomers are colored green and yellow in both cases. Top, reported asymmetric unit for DBL6ɛ; middle, reorganized dimer based solely on crystal symmetry; bottom. RII-PvDBP dimer. () Overlay of RII-PvDBP (green) and DBL6ɛ (brown) reveals critical binding residues for each protein superpose well (Lys273 and Arg274 from RII-PvDBP, and Lys2392 and Lys2395 from DBL6ɛ). * Figure 5: PvDBP binds DARC via a model of receptor-mediated ligand-dimerization. PvDBP exists as an equilibrium of monomers and dimers that is shifted to dimerization upon receptor binding. RII-PvDBP monomers are in green and yellow. The P. vivax membrane is in black, and the reticulocyte membrane is in red. Flat lines represent portions of PvDBP not in the crystal structure. The DARC homodimer is represented by the crystal structure of the homodimeric membrane-spanning region of a related GPCR, CXCR4 (ref. 40), in dark/light red. DARC1–60 is shown as a flat line. Two PvDBP molecules bind two DARC molecules, as indicated by our stoichiometry measurements. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3RRC * 3RRC Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, Missouri, USA. * Joseph D Batchelor, * Jacob A Zahm & * Niraj H Tolia Contributions J.D.B. performed functional assays, SAXS data analysis, AUC studies, ITC studies and structure analyses. J.A.Z. cloned, purified and crystallized RII-PvDBP. N.H.T. designed the study, analyzed SAXS data, collected, processed and refined X-ray data, and analyzed the structure. All authors were involved in writing the paper, discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Niraj H Tolia Author Details * Joseph D Batchelor Search for this author in: * NPG journals * PubMed * Google Scholar * Jacob A Zahm Search for this author in: * NPG journals * PubMed * Google Scholar * Niraj H Tolia Contact Niraj H Tolia 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 (963K) Supplementary Figures 1–7 and Supplementary Methods Additional data Entities in this article * C-X-C chemokine receptor type 4 CXCR4 Homo sapiens * View in UniProt * View in Entrez Gene * C-C chemokine receptor type 5 CCR5 Homo sapiens * View in UniProt * View in Entrez Gene * Envelope glycoprotein gp160 env Human immunodeficiency virus type 1 group M subtype B (isolate HXB2) * View in UniProt * View in Entrez Gene * Duffy receptor PVX_110810 Plasmodium vivax (strain Salvador I) * View in UniProt * View in Entrez Gene * Erythrocyte membrane protein 1, PfEMP1 VAR Plasmodium falciparum (isolate 3D7) * View in UniProt * View in Entrez Gene * Putative erythrocyte binding protein EBL-1 Plasmodium falciparum * View in UniProt * Micronemal protein MIC2 Toxoplasma gondii * View in UniProt * MIC3 microneme protein TGME49_119560 Toxoplasma gondii * View in UniProt * View in Entrez Gene * Erythrocyte-binding antigen 175 Plasmodium falciparum (isolate Camp / Malaysia) * View in UniProt * Duffy antigen/chemokine receptor DARC Homo sapiens * View in UniProt * View in Entrez Gene * Erythrocyte binding antigen-181 eba-181 Plasmodium falciparum (isolate 3D7) * View in UniProt * View in Entrez Gene * Erythrocyte binding antigen-140 eba-140 Plasmodium falciparum (isolate 3D7) * View in UniProt * View in Entrez Gene - Crystal structure of γ-tubulin complex protein GCP4 provides insight into microtubule nucleation
- Nat Struct Mol Biol 18(8):915-919 (2011)
Nature Structural & Molecular Biology | Article Crystal structure of γ-tubulin complex protein GCP4 provides insight into microtubule nucleation * Valérie Guillet1, 2, 6 * Martine Knibiehler3, 6 * Lynn Gregory-Pauron1, 2, 6 * Marie-Hélène Remy3 * Cécile Chemin3 * Brigitte Raynaud-Messina3 * Cécile Bon1, 2 * Justin M Kollman4, 5 * David A Agard4, 5 * Andreas Merdes3 * Lionel Mourey1, 2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:915–919Year published:(2011)DOI:doi:10.1038/nsmb.2083Received24 February 2011Accepted03 May 2011Published online03 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 Microtubule nucleation in all eukaryotes involves γ-tubulin small complexes (γTuSCs) that comprise two molecules of γ-tubulin bound to γ-tubulin complex proteins (GCPs) GCP2 and GCP3. In many eukaryotes, multiple γTuSCs associate with GCP4, GCP5 and GCP6 into large γ-tubulin ring complexes (γTuRCs). Recent cryo-EM studies indicate that a scaffold similar to γTuRCs is formed by lateral association of γTuSCs, with the C-terminal regions of GCP2 and GCP3 binding γ-tubulin molecules. However, the exact role of GCPs in microtubule nucleation remains unknown. Here we report the crystal structure of human GCP4 and show that its C-terminal domain binds directly to γ-tubulin. The human GCP4 structure is the prototype for all GCPs, as it can be precisely positioned within the γTuSC envelope, revealing the nature of protein-protein interactions and conformational changes regulating nucleation activity. View full text Figures at a glance * Figure 1: The crystal structure of GCP4 reveals a previously undescribed fold. () Topology diagram (left) and ribbon representation (right). The first helix bundle is in light blue, the second in purple, the third in orange, the fourth in light pink and the fifth in blue. All structural elements excluded from helix bundles are in green. Helices and beta strands are numbered. Stretches of missing residues are represented by dashed lines (left) and by colored spheres (right). Residues preceding and following missing loops are labeled. () Ribbon representation colored according to sequence similarity over orthologous GCP4 proteins as shown in Supplementary Figure 1. Residues with similarity <80% are in white; conserved areas with similarity in the range 80–100% are colored light red to bright red. * Figure 2: GCP4 binds to γ-tubulin through its C-terminal domain. () View down the convex face and side view of the molecular surface of GCP4. Conserved residues are colored as in Figure 1b. Nonconserved residues of the N- and C-terminal domains are colored light- and dark gray, respectively. (–) Co-transcription–translation of V5-tagged GCP4 and Flag-tagged γ-tubulin in reticulocyte lysate, followed by immunoprecipitation with anti-Flag affinity beads. Immunoblots of the eluted fractions from beads rinsed with HEPES buffer, followed by consecutive treatments with 0.5 M NaCl, 0.1% NP40, RIPA buffer, 0.1% SDS and gel loading buffer according to Laemmli22 are shown, probed with antibody against γ-tubulin () or against the V5 tag (–). Full-length () and deletion constructs (–) of GCP4 were tested as indicated on the figure. Amino acid, aa. * Figure 3: A molecular model of γTuSC. () Models of GCP2 and GCP3 generated from the GCP4 crystal structure and the γ-tubulin crystal structure were manually fit into the cryo-EM γTuSC reconstruction. The inset shows the region of GCP3 previously shown by EM to act as a hinge, corresponding to the interface between helical bundles 3 (blue) and 4 (cyan); the position of Trp460 in GCP4 is indicated with a red sphere. Empty regions of the EM map include density corresponding to an insert in GCP2 (black arrowhead) and the density of a bound adaptor protein (Spc110) in the EM structure (gray arrowheads). () The EM map sharpened with a 500-Å2B-factor has α-helical features in good agreement with bundles 2 and 3 of GCP3. () Semi-transparent molecular surface of the C-terminal domain of GCP4 colored according to sequence similarity over hGCPs as shown in Supplementary Figure 1. Enlarged stereo view (right) of the conserved cavity is colored with respect to atom type (C, gray; N, blue; O, red) and sequence conservatio! n of important residues (given in the hGCP2-hGCP3-hGCP4-hGCP5-hGCP6 order) that delineate the cavity. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3RIP * 3RIP Referenced accessions Protein Data Bank * 1Z5V * 1Z5V Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Valérie Guillet, * Martine Knibiehler & * Lynn Gregory-Pauron Affiliations * Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, Toulouse, France. * Valérie Guillet, * Lynn Gregory-Pauron, * Cécile Bon & * Lionel Mourey * Université de Toulouse, Université Paul Sabatier, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France. * Valérie Guillet, * Lynn Gregory-Pauron, * Cécile Bon & * Lionel Mourey * Centre de Recherche en Pharmacologie-Santé, Centre National de la Recherche Scientifique–Pierre Fabre, Toulouse, France. * Martine Knibiehler, * Marie-Hélène Remy, * Cécile Chemin, * Brigitte Raynaud-Messina & * Andreas Merdes * Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, California, USA. * Justin M Kollman & * David A Agard * Keck Advanced Microscopy Center, University of California, San Francisco, California, USA. * Justin M Kollman & * David A Agard Contributions V.G. helped optimize protein production and purification, grew the crystals and conducted diffraction data collection, analyzed the structure, prepared figures and participated in manuscript writing. M.K. optimized native and SeMet-labeled protein production and purification. L.G.-P. participated in data processing and did structure determination and refinement, analyzed the structure and helped prepare tables and figures. M.-H.R. made the constructs, produced and purified the proteins, carried out Flag pulldown experiments and prepared figures. C.C. made the constructs and carried out Flag pulldown experiments. B.R.-M. did initial purification studies. C.B. participated in protein characterization. J.M.K. analyzed structure-function relationships, carried out the fitting into the cryo-EM map of γTuSC and prepared figures. D.A.A. analyzed the data and revised the manuscript. A.M. devised the experiments, designed figures and wrote the manuscript. L.M. devised the experiment! s, participated in diffraction data collection, analyzed the structure, designed tables and figures and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Andreas Merdes or * Lionel Mourey Author Details * Valérie Guillet Search for this author in: * NPG journals * PubMed * Google Scholar * Martine Knibiehler Search for this author in: * NPG journals * PubMed * Google Scholar * Lynn Gregory-Pauron Search for this author in: * NPG journals * PubMed * Google Scholar * Marie-Hélène Remy Search for this author in: * NPG journals * PubMed * Google Scholar * Cécile Chemin Search for this author in: * NPG journals * PubMed * Google Scholar * Brigitte Raynaud-Messina Search for this author in: * NPG journals * PubMed * Google Scholar * Cécile Bon Search for this author in: * NPG journals * PubMed * Google Scholar * Justin M Kollman Search for this author in: * NPG journals * PubMed * Google Scholar * David A Agard Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Merdes Contact Andreas Merdes Search for this author in: * NPG journals * PubMed * Google Scholar * Lionel Mourey Contact Lionel Mourey 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 (19M) Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Discussion Additional data Entities in this article * Gamma-tubulin complex component 4 TUBGCP4 Homo sapiens * View in UniProt * View in Entrez Gene * Spindle pole body component 110 SPC110 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Putative spindle pole body protein AT5G17410 Arabidopsis thaliana * View in UniProt * View in Entrez Gene * Gamma-tubulin complex component 3 TUBGCP3 Homo sapiens * View in UniProt * View in Entrez Gene * Gamma-tubulin complex component 2 TUBGCP2 Homo sapiens * View in UniProt * View in Entrez Gene * Spindle pole body component SPC98 SPC98 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Spindle pole body component SPC97 SPC97 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Gamma-tubulin complex component 6 TUBGCP6 Homo sapiens * View in UniProt * View in Entrez Gene * Gamma-tubulin complex component 5 TUBGCP5 Homo sapiens * View in UniProt * View in Entrez Gene - SUMOylation regulates telomere length homeostasis by targeting Cdc13
- Nat Struct Mol Biol 18(8):920-926 (2011)
Nature Structural & Molecular Biology | Article SUMOylation regulates telomere length homeostasis by targeting Cdc13 * Lisa E Hang1, 2, 5 * Xianpeng Liu1, 4, 5 * Iris Cheung3, 4 * Yan Yang1, 4 * Xiaolan Zhao1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:920–926Year published:(2011)DOI:doi:10.1038/nsmb.2100Received03 June 2011Accepted15 June 2011Published online10 July 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 Telomere length homeostasis is an important aspect of telomere biology. Here, we show that SUMOylation limits telomere length and targets multiple telomere proteins in Saccharomyces cerevisiae. A main target is Cdc13, which both positively and negatively regulates telomerase and confers end protection. We demonstrate that Cdc13 SUMOylation restrains telomerase functions by promoting Cdc13 interaction with the telomerase inhibitor Stn1 without affecting end protection. Mutation of the Cdc13 SUMOylation site (cdc13-snm) lengthens telomeres and reduces the Stn1 interaction, whereas Cdc13-SUMO fusion has the opposite effects. cdc13-snm's effect on telomere length is epistatic with stn1, but not with yku70, tel1 or est1 alleles, and is suppressed by Stn1 overexpression. Cdc13 SUMOylation peaks in early-mid S phase, prior to its known Cdk1-mediated phosphorylation, and the two modifications act antagonistically, suggesting that the opposite roles of Cdc13 in telomerase regulation ! can be separated temporally and regulated by distinct modifications. View full text Figures at a glance * Figure 1: Defects in SUMOylation enzymes lead to longer telomeres, and multiple telomere proteins are SUMOylated. (–) ubc9 mutants () and two SUMO E3 double mutants (), but not SUMO E3 single mutants (), show longer telomeres. ubc9-MYC and ubc9-HA are the Myc- and hemagglutinin (HA)-tagged UBC9 genes, respectively. mms21 mutants contain two point mutations in their SUMO E3 domain25. () Rap1, Yku70, Cdc13, Pif1 and Yku80 are SUMOylated. Proteins were tagged with TAP (not labeled) or Myc and were immunopurified before (−) and after (+) MMS treatment. Unmodified proteins were detected on immunoblots by anti-ProA or anti-Myc antibodies (below) and SUMOylated forms of the proteins by anti-SUMO antibody (above). Asterisks label the positions of SUMOylated proteins before MMS treatment. The occasional difference in signals on the anti-tag blots reflects variation in loading rather than protein levels. () SUMOylation of Rap1, Yku70, Cdc13 and Pif1, but not Rad52, increases at 37 °C. * Figure 2: SUMOylation of Cdc13 at a single lysine peaks in early to mid S phase. () Cdc13 SUMOylation peaks in early to mid S phase. Alpha-factor–arrested G1 cells (0 min) containing Cdc13-TAP were released and samples at indicated time points were examined for protein SUMOylation (left) and by FACS analysis (right). The position of SUMOylated Cdc13 is labeled as Cdc13-S. () Schematic map of Cdc13. Stn1- and Pol1-interacting domains as well as telomerase recruitment (RD) and DNA binding (DBD) domains are labeled12, 32, 33, 34, 35, 36. The dot represents the Cdk1 phosphorylation site. The SUMOylation consensus site and the position of the SUMOylated lysine are depicted. () K909R abolishes Cdc13 SUMOylation. Wild-type Cdc13 (WT) or Cdc13-K909R proteins tagged with TAP were examined as in Figure 1d. () cdc13-snm does not affect protein levels. Protein extracts before (−) and after (+) MMS treatment were examined by immunoblots using anti-ProA antibody. Cdc13 and Cdc13-snm were tagged with TAP. Equal loading is shown by amido black staining. * Figure 3: cdc13-snm leads to longer telomeres in a telomerase-dependent manner. () cdc13-snm cells show increased telomere length. () Deleting the telomerase component TLC1 abolishes telomere lengthening caused by cdc13-snm. () cdc13-snm and tlc1Δ show no synthetic defects in senescence. Senescence experiments were conducted by repeatedly streaking strains (number of times streaked is indicated for each). * Figure 4: cdc13-snm weakens Stn1 interaction and increases telomere length in yku70Δ, tel1Δ and EST1-myc cells. (,) Cdc13-snm affects Stn1 interaction in yeast two-hybrid assays. Two-hybrid strain containing pairs of indicated GTPase-binding domain (GBD) and glutamic acid decarboxylase (GAD) plasmids were grown on medium lacking leucine and tryptophan (–LEU–TRP). The activation of reporters was scored by replica plating onto –LEU–TRP–ADE and –LEU–TRP–HIS media () and by assaying the activity of β-galactosidase (). Cdc13-snm specifically weakens interaction with Stn1 (P = 0.0018) but not with Pol1 (). Error bars represent s.d. (–) cdc13-snm leads to longer telomeres in yku70Δ, tel1Δ and EST-myc cells. EST-myc was generated by tagging Est1 at its N terminus with 13MYC. * Figure 5: stn1 alleles are epistatic to cdc13-snm and specifically enhance Cdc13 SUMOylation. (,) stn1 alleles are epistatic to cdc13-snm. The double mutants of cdc13-snm with stn1-myc () or stn1-ΔC199 () have telomere lengths similar to those of the stn1 single mutants. (,) stn1 alleles enhance the SUMOylation of Cdc13 but not that of Rap1 and Yku70. The effects of stn1-myc (-myc) and stn1ΔC199 (ΔC) on SUMOylation of Cdc13-TAP () and of Rap1 and Yku70 () were examined. (,) Cdc13-TAP SUMOylation is not altered in rif1Δ () or in yku70Δ, tel1Δ and EST1-myc cells (). * Figure 6: CDC13-SUMO leads to shorter telomeres and enhanced Stn1 interaction, and the two modifications of Cdc13Cdc13 act antagonistically. () CDC13-SUMO leads to shorter telomeres and cdc13-T308A-SUMO results in a greater degree of telomere shortening than either cdc13-T308A or CDC13-SUMO. () CDC13-SUMO fusion enhances Stn1 interaction. Untagged and Myc-tagged Stn1 cells both contain Cdc13-HA. Protein extracts from these cells were co-immunoprecipitated with anti-Myc antibody, and Cdc13 and Cdc13-SUMO were examined in the immunoprecipitated fraction with anti-HA antibody. The numbers indicate the relative amounts of co-purified Cdc13 in wild-type and CDC13-SUMO strains based on the average of four trials (mean ± s.d.). Cdc13 and Cdc13-SUMO input levels are similar (data not shown). (,) Abolition of Cdk1-mediated phosphorylation of Cdc13 does not affect its SUMOylation, and vice versa. HA-tagged wild-type and mutant Cdc13 were examined for SUMOylation () and phosphorylation () after immunoprecipitation. () Lack of Cdk1-mediated Cdc13 phosphorylation and lack of Cdc13 SUMOylation affect telomere length in an ant! agonistic manner. Telomere length was examined in wild-type (WT) and cdc13 mutant cells, and telomere length of cdc13-T308A snm cells was found to be inbetween those of cdc13-T308A and cdc13-snm cells. () Models for the roles of the two protein modifications on Cdc13. See text for details. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Lisa E Hang & * Xianpeng Liu Affiliations * Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA. * Lisa E Hang, * Xianpeng Liu, * Yan Yang & * Xiaolan Zhao * Programs in Biochemistry, Cell and Molecular Biology, Weill Graduate School of Medical Sciences, Cornell University, New York, New York, USA. * Lisa E Hang & * Xiaolan Zhao * Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA. * Iris Cheung * Present addresses: Department of Biochemistry and Molecular Biology, Louisiana State University, Health Sciences Center, Shreveport, Louisiana, USA (X.L.); Eurofins MWG Operon, Huntsville, Alabama, USA (I.C.); New York University Medical School, New York, New York, USA (Y.Y.). * Xianpeng Liu, * Iris Cheung & * Yan Yang Contributions X.Z. directed the study. X.Z., X.L. and L.E.H. designed the experiments. X.L., L.E.H., Y.Y. and I.C. carried out the experiments. All authors were involved in data analysis. The manuscript was prepared by X.Z. with the assistance of L.E.H. and X.L. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Xiaolan Zhao Author Details * Lisa E Hang Search for this author in: * NPG journals * PubMed * Google Scholar * Xianpeng Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Iris Cheung Search for this author in: * NPG journals * PubMed * Google Scholar * Yan Yang Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaolan Zhao Contact Xiaolan Zhao Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–6 and Supplementary Table 1 Additional data Entities in this article * Telomere length regulator protein RIF1 RIF1 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 * DNA repair and recombination protein PIF1 PIF1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Protein RIF2 RIF2 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 * Protein STN1 STN1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Telomere elongation protein EST1 EST1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * 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 * E3 SUMO-protein ligase SIZ1 SIZ1 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 * SUMO-conjugating enzyme UBC9 UBC9 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Telomere length regulation protein TEN1 TEN1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Cell division control protein 13 CDC13 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * 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 * DNA-binding protein RAP1 RAP1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * E3 SUMO-protein ligase MMS21 MMS21 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * E3 SUMO-protein ligase SIZ2 NFI1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA polymerase alpha catalytic subunit A POL1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Telomerase RNA TLC1 Saccharomyces cerevisiae S288c * View in Entrez Gene * DNA repair and recombination protein RAD52 RAD52 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene - Complexin cross-links prefusion SNAREs into a zigzag array
- Nat Struct Mol Biol 18(8):927-933 (2011)
Nature Structural & Molecular Biology | Article Complexin cross-links prefusion SNAREs into a zigzag array * Daniel Kümmel1 * Shyam S Krishnakumar1 * Daniel T Radoff1, 2 * Feng Li1 * Claudio G Giraudo1 * Frederic Pincet1, 3 * James E Rothman1 * Karin M Reinisch1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:927–933Year published:(2011)DOI:doi:10.1038/nsmb.2101Received09 February 2011Accepted19 May 2011Published online24 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 Complexin prevents SNAREs from releasing neurotransmitters until an action potential arrives at the synapse. To understand the mechanism for this inhibition, we determined the structure of complexin bound to a mimetic of a prefusion SNAREpin lacking the portion of the v-SNARE that zippers last to trigger fusion. The 'central helix' of complexin is anchored to one SNARE complex, while its 'accessory helix' extends away at ~45° and bridges to a second complex, occupying the vacant v-SNARE binding site to inhibit fusion. We expected the accessory helix to compete with the v-SNARE for t-SNARE binding but found instead that the interaction occurs intermolecularly. Thus, complexin organizes the SNAREs into a zigzag topology that, when interposed between the vesicle and plasma membranes, is incompatible with fusion. View full text Figures at a glance * Figure 1: Structure of the prefusion CPX–SNARE complex. VAMP2 (residues 29–60), blue; syntaxin (residues 190–250), yellow; SNAP25 (N-terminal SNARE motif, residues 10–74), lime and (C-terminal SNARE motif, residues 141–203), green; CPX (residues 26–73), cyan. (,) Model of the truncated SNARE complex without () and with () CPX bound. CPXcen, cyan; CPXacc, pale cyan. A dashed arrow indicates syntaxin membrane anchor. () Comparison of pre- and postfusion CPX–SNARE complexes, with postfusion CPX in magenta (CPXacc is pale magenta, PDB 1KIL). () Top and side views of the zigzag array of postfusion CPX–SNARE complexes observed in crystals. SNAREpins are related by 180° rotation and translation along the zigzag midline, so that on different sides of the midline, the linkers that connect syntaxins and VAMPs to their transmembrane helices are on opposite sides of the zigzag plane. * Figure 2: Interacting surfaces of CPXacc and the t-SNAREs. () Interacting residues of scCPX are labeled in left panels; the binding site on the t-SNARE is outlined as gray patch and labeled on right panels. () For scCPX-F34M, CPXacc can bind to the t-SNARE groove as in or as shown here. () Sequence of the accessory helix of wild-type (WT) CPX and the nonclamping (nc) and superclamp (sc) mutants. Residues of CPX interacting with the t-SNARE in the crystal structures are boxed. The side chain of Lys26 is disordered in our structure, but functional data19 suggest that it has a role in clamping. It may interact with the VAMP2 C terminus that is absent in our structure. * Figure 3: Characterization of the interaction of CPXacc with SNARE complexes by isothermal titration calorimetry. () A groove in the t-SNARE is a second binding site for CPX distinct from the central helix binding site. When the central helix binding site on the SNARE complex is blocked, CPX still binds to the SNARE complex once the C-terminal half of VAMP2 is removed from the prefusion SNARE mimetic. () Binding to the t-SNARE groove is mediated by CPXacc. Mutations in the accessory helix of CPX modulate the binding affinity to the t-SNARE positively (sc CPX) or negatively (nc CPX), as expected from the crystal structure. * Figure 4: FRET experiments probing CPX orientation in pre- and postfusion CPX–SNARE complexes. () Superposition of pre- and postfusion CPX–SNARE complexes, where prefusion CPX is depicted in cyan and postfusion CPX in pale cyan. As indicated (magenta spheres), SNAP25 was labeled with stilbene at position 193, and CPX was labeled with bimane at positions 31 or 38. () Fluorescence emission spectra of stilbene only (black) and stilbene- or bimane-labeled CPX–SNARE complexes containing VAMP2 (residues 25–96, red), VAMP2-Δ60 (residues 25–60, cyan), or VAMP2-4X (residues 25–96 with mutations L70D, A74R, A81D and L84D to preclude zippering of the VAMP2 C terminus, blue). CPX is labeled with bimane at residue 38. () As in , but CPX is labeled with bimane at residue 31. These data were used to calculate the distances shown in Supplementary Table 3. () FRET of a flexible CPX mutant (CPX-GPGP) in comparison to wild-type (WT) CPX when bound to prefusion (VAMP2-Δ60) or postfusion (VAMP2) SNARE complexes. When the accessory helix is uncoupled from the central helix by a! helix-breaking GPGP insertion, there is a complete loss of FRET signal with both SNARE complexes, different from the partial change in FRET observed with intact CPX. Thus, it is unlikely that differences between the FRET signals observed with intact CPX are due to random motion in CPXacc. * Figure 5: Effects of CPX and VAMP2 mutations on clamping in cell-cell fusion assays. () Mutational analysis of CPX accessory helix mutations in the cell-cell fusion assay. Experiments were carried out in the presence of CPX only (CPX) or, additionally, phosphatidylinositol-specific phospholipase C, Ca2+ and SYT (CPX/PI-PLC/Ca/SYT). Results are mean ± s.e.m. of three independent experiments. () Mapping of the mutational analysis of the CPXacc–t-SNARE interface. CPXacc is shown, with the surface that interacts with the t-SNARE in the crystal structures outlined in black. Mutations in CPX that affect clamping positively (green) or negatively (red) are at the interface. Mutations that do not affect clamping (blue) are on the opposite side of CPX. () Location of the helix-breaking mutations (magenta) between central and accessory helix in the CPX–SNARE prefusion crystal structure. Results are mean ± s.e.m. of three independent experiments. * Figure 6: Molecular models for CPX clamping. () Model for the clamp at the synapse. CPX–SNARE complexes with half-zippered VAMP2 are cross-linked by CPX into a zigzag topology incompatible with fusion (see text). The plane of the zigzag is normal to the vertical direction. For clarity, only two of the CPX–SNARE complexes in the zigzag are shown. Palmitoylation on SNAP25 is indicated and restrains the distance between the CPX–SNARE zigzag and the plasma membrane (PM). The distance between the zigzag plane and the vesicle (SV) must be less than ~110 Å, the maximum distance spanned by the v-SNARE linker. The calcium sensor synaptotagmin (gray with Ca2+-binding loops in orange), which relieves CPX clamping, is accommodated by this model and is positioned based on FRET analysis45. Its Ca2+-binding loops are juxtaposed to the vesicle membrane, which is rich in anionic lipids like phosphatidylserine, and is well positioned for interactions with this membrane in response to Ca2+ stimulus. () Model of the CPX–SNARE ass! embly in the clamped state when the fusion pore is 'closed' (left). The fusion pore can open only once the zigzag clamped array has disassembled (right). Complexes in the 'open' state are modeled on PDB 1KIL. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3RK2 * 3RK3 * 3RL0 * 3RK2 * 3RK3 * 3RL0 Referenced accessions Protein Data Bank * 1KIL * 1KIL Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA. * Daniel Kümmel, * Shyam S Krishnakumar, * Daniel T Radoff, * Feng Li, * Claudio G Giraudo, * Frederic Pincet, * James E Rothman & * Karin M Reinisch * Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA. * Daniel T Radoff * Laboratoire de Physique Statistique, Unité Mixte de Recherche 8550, Centre National de la Recherche Scientifique associée aux Universités Paris VI et Paris VII, Ecole Normale Supérieure, Paris, France. * Frederic Pincet Contributions D.K. coordinated the experiments in this paper, was responsible for structure analysis and designed constructs for the functional analyses. S.S.K. and D.T.R. conducted the FRET experiments; F.L. conducted the ITC analysis and C.G.G. carried out the cell-cell fusion experiments. F.P. contributed to the analysis of the FRET and ITC data. D.K., J.E.R. and K.M.R. analyzed data and wrote this manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * James E Rothman or * Karin M Reinisch Author Details * Daniel Kümmel Search for this author in: * NPG journals * PubMed * Google Scholar * Shyam S Krishnakumar Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel T Radoff Search for this author in: * NPG journals * PubMed * Google Scholar * Feng Li Search for this author in: * NPG journals * PubMed * Google Scholar * Claudio G Giraudo Search for this author in: * NPG journals * PubMed * Google Scholar * Frederic Pincet Search for this author in: * NPG journals * PubMed * Google Scholar * James E Rothman Contact James E Rothman Search for this author in: * NPG journals * PubMed * Google Scholar * Karin M Reinisch Contact Karin M Reinisch 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 1–4, Supplementary Tables 1–3 and Supplementary Methods Additional data Entities in this article * Vesicle-associated membrane protein 2 VAMP2 Homo sapiens * View in UniProt * View in Entrez Gene * Complexin-1 CPLX1 Homo sapiens * View in UniProt * View in Entrez Gene * Synaptosomal-associated protein 25 SNAP25 Homo sapiens * View in UniProt * View in Entrez Gene * 1-phosphatidylinositol phosphodiesterase Bacillus cereus * View in UniProt * Syntaxin-1A Stx1a Rattus norvegicus * View in UniProt * View in Entrez Gene - A conformational switch in complexin is required for synaptotagmin to trigger synaptic fusion
- Nat Struct Mol Biol 18(8):934-940 (2011)
Nature Structural & Molecular Biology | Article A conformational switch in complexin is required for synaptotagmin to trigger synaptic fusion * Shyam S Krishnakumar1, 4 * Daniel T Radoff1, 2, 4 * Daniel Kümmel1 * Claudio G Giraudo1 * Feng Li1 * Lavan Khandan1 * Stephanie Wood Baguley1 * Jeff Coleman1 * Karin M Reinisch1 * Frederic Pincet1, 3 * James E Rothman1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:934–940Year published:(2011)DOI:doi:10.1038/nsmb.2103Received09 February 2011Accepted19 May 2011Published online24 July 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 The crystal structure of complexin bound to a prefusion SNAREpin mimetic shows that the accessory helix extends away from the SNAREpin in an 'open' conformation, binding another SNAREpin and inhibiting its assembly, to clamp fusion. In contrast, the accessory helix in the postfusion complex parallels the SNARE complex in a 'closed' conformation. Here we use targeted mutations, FRET spectroscopy and a functional assay that reconstitutes Ca2+-triggered exocytosis to show that the conformational switch from open to closed in complexin is needed for synaptotagmin-Ca2+ to trigger fusion. Triggering fusion requires the zippering of three crucial aspartate residues in the switch region (residues 64–68) of v-SNARE. Conformational switching in complexin is integral to clamp release and is probably triggered when its accessory helix is released from its trans-binding to the neighboring SNAREpin, allowing the v-SNARE to complete zippering and open a fusion pore. View full text Figures at a glance * Figure 1: Zippering of one turn of VAMP2 helix triggers the switch in CPX position. () Superposition of the structures of the pre- and postfusion CPX–SNARE complexes21, 25, showing the FRET label positions. Syntaxin1 (residues 190–250) is in yellow, SNAP25 N-terminal SNARE motif (residues 10–74) is in pale green, SNAP25 C-terminal SNARE motif (residues 141–203) is in green and VAMP2 is in blue (residues 25–60 dark blue; residues 61–96 in light blue). The switch residues (Asp64, Asp65 and Asp68) are marked in red. The complexin (residues 26–73) in the prefusion complex is in cyan, and in the postfusion complex is in light cyan. The FRET label positions, residue 193 on SNAP25 and residue 38 on complexin, are marked as magenta spheres. The sequence of the C-terminal hydrophobic layer of VAMP2 (residues 57–90) with the C-terminal truncations tested in this paper (denoted by the residue number) is also shown. The black arrow in the postfusion structure references CPX residue 48, the demarcation line between CPXcen and CPXacc. () FRET experiments ! with C-terminal truncations of VAMP2. Fluorescence emission spectra of stilbene- and bimane-labeled CPX–SNARE complexes containing VAMP2-60 (residues 25–60, orange), VAMP2-65 (residues 25–65, green), VAMP2-69 (residues 25–69, purple), VAMP2-73 (residues 25–73, blue), VAMP2-77 (residues 25–77, red) and VAMP2 (residues 1–96, olive). A representative emission spectrum of a stilbene (donor)-only CPX–SNARE complex is shown in black. The donor-only spectrum was identical in all CPX–SNARE complexes. * Figure 2: Aspartate residues 64, 65 and 68 on VAMP2 mediate the switch in CPX position. () Hydrogen bonding and salt bridge interactions between the switch aspartate residues 64, 65 and 68 with CPXcen helix in the postfusion complex25. () Complexin adopts an open conformation in CPX–SNARE complexes containing either VAMP-3xDA or VAMP-4X. Donor fluorescence at 410 nm (normalized to a donor-only sample) for stilbene-bimane–labeled CPX–SNARE complexes containing VAMP2-D64A, VAMP2-D65A, VAMP2-D68A, VAMP-3xDA or VAMP-4X is shown. (The raw fluorescence emission curves are shown in Supplementary Fig. 3.) The donor fluorescence (at 410 nm) for VAMP2-60 (open) and VAMP2 (closed) are shown for comparison. Averages and s.d. for three or four independent experiments are shown. * Figure 3: Interaction of CPXcen with aspartate residues 64, 65 and 68 on VAMP2 provides thermodynamic driving force for the switch. Calorimetric titrations of superclamp CPX (scCPX; residues 1–134 with D27L E34F R37A mutations) into assembled SNARE complexes containing t-SNAREs and either wild-type (WT) VAMP2 (blue triangles), VAMP2-3xDA (red squares) or VAMP-3xDA with the CPXcen binding site blocked by CPX 48–134 (black circles). The solid lines represent the best fit to the corresponding data points using a nonlinear least-squares fit with a one-set-of-sites model. The results of the fits are given in Table 2. All experiments were conducted in triplicate at 37 °C, and a representative thermogram is shown. * Figure 4: The switch in CPXacc position is necessary for synaptotagmin-Ca2+ to trigger fusion. () Clamping of SNARE-mediated fusion by CPX and the reversal of the clamp by synaptotagmin-Ca2+ in the presence of phosphatidylinositol-specific phospholipase-C (PI-PLC) in wild-type (WT) VAMP2, VAMP2-D64A, VAMP2-D68A and VAMP-3xDA as measured in a cell-cell fusion assay. The effect of the superclamp CPX (scCPX; CPX D27L, E34F, R37A) on wild-type VAMP2 is shown for comparison. () Kinetics of the reversal of the CPX clamp by synaptotagmin-Ca2+. The cell fusion recovery was carried out at 1 mM free Ca2+, and the samples were fixed at the indicated time point after the addition of Ca2+. Averages and s.d. of two or three independent experiments are shown. * Figure 5: Perturbation of a single SNARE complex in the zigzag array should be sufficient to rapidly disassemble the clamp in response to neuronal stimulus. A small perturbation of one CPX–SNARE complex in the clamped zigzag array would eliminate interactions with both of its neighbors in the array. For example, a disruption of complex 3 would eliminate interaction with complexes 2 and 4. Therefore, if one set of the CPXacc-SNAREpin interactions were to be perturbed by synaptotagmin-Ca2+, then VAMP2 could zipper up, and the entire zigzag array would disassemble very rapidly, releasing the clamp and triggering fusion. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Shyam S Krishnakumar & * Daniel T Radoff Affiliations * Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA. * Shyam S Krishnakumar, * Daniel T Radoff, * Daniel Kümmel, * Claudio G Giraudo, * Feng Li, * Lavan Khandan, * Stephanie Wood Baguley, * Jeff Coleman, * Karin M Reinisch, * Frederic Pincet & * James E Rothman * Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA. * Daniel T Radoff * Laboratoire de Physique Statistique, Unité Mixte de Recherche 8550, Centre National de la Recherche Scientifique associée aux Université Paris VI et Paris VII, Ecole Normale Supérieure, Paris, France. * Frederic Pincet Contributions S.S.K., D.T.R. and D.K. did the mutagenesis and protein purification, S.S.K. and D.T.R. carried out the fluorescence measurements, F.L. took the ITC measurements and C.G.G. did the cell-cell fusion assay. L.K., S.B. and J.C. provided technical assistance. Results were analyzed and discussed by all authors. The manuscript was prepared by S.S.K., D.T.R., K.M.R., F.P. and J.E.R. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * James E Rothman Author Details * Shyam S Krishnakumar Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel T Radoff Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Kümmel Search for this author in: * NPG journals * PubMed * Google Scholar * Claudio G Giraudo Search for this author in: * NPG journals * PubMed * Google Scholar * Feng Li Search for this author in: * NPG journals * PubMed * Google Scholar * Lavan Khandan Search for this author in: * NPG journals * PubMed * Google Scholar * Stephanie Wood Baguley Search for this author in: * NPG journals * PubMed * Google Scholar * Jeff Coleman Search for this author in: * NPG journals * PubMed * Google Scholar * Karin M Reinisch Search for this author in: * NPG journals * PubMed * Google Scholar * Frederic Pincet Search for this author in: * NPG journals * PubMed * Google Scholar * James E Rothman Contact James E Rothman Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (725K) Supplementary Figures 1–4 Additional data Entities in this article * Vesicle-associated membrane protein 2 VAMP2 Homo sapiens * View in UniProt * View in Entrez Gene * Complexin-1 CPLX1 Homo sapiens * View in UniProt * View in Entrez Gene * Synaptosomal-associated protein 25 SNAP25 Homo sapiens * View in UniProt * View in Entrez Gene * 1-phosphatidylinositol phosphodiesterase Bacillus cereus * View in UniProt * Syntaxin-1A Stx1a Rattus norvegicus * View in UniProt * View in Entrez Gene - Complexin activates and clamps SNAREpins by a common mechanism involving an intermediate energetic state
- Nat Struct Mol Biol 18(8):941-946 (2011)
Nature Structural & Molecular Biology | Article Complexin activates and clamps SNAREpins by a common mechanism involving an intermediate energetic state * Feng Li1, 2 * Frédéric Pincet1, 2 * Eric Perez2 * Claudio G Giraudo1 * David Tareste1, 3 * James E Rothman1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:941–946Year published:(2011)DOI:doi:10.1038/nsmb.2102Received09 February 2011Accepted16 June 2011Published online24 July 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 The core mechanism of intracellular vesicle fusion consists of SNAREpin zippering between vesicular and target membranes. Recent studies indicate that the same SNARE-binding protein, complexin (CPX), can act either as a facilitator or as an inhibitor of membrane fusion, constituting a controversial dilemma. Here we take energetic measurements with the surface force apparatus that reveal that CPX acts sequentially on assembling SNAREpins, first facilitating zippering by nearly doubling the distance at which v- and t-SNAREs can engage and then clamping them into a half-zippered fusion-incompetent state. Specifically, we find that the central helix of CPX allows SNAREs to form this intermediate energetic state at 9–15 nm but not when the bilayers are closer than 9 nm. Stabilizing the activated-clamped state at separations of less than 9 nm requires the accessory helix of CPX, which prevents membrane-proximal assembly of SNAREpins. View full text Figures at a glance * Figure 1: CPX affects the structural-energetic landscape of SNAREpins as they assemble across membranes. (–) Interaction energy versus distance profile of SNAREpins in the absence (black) or the presence (purple) of CPX at various concentrations (squares, approach; circles, separation). The interaction profiles between SNARE bilayers have been normalized to the surface density of SNAREs in the apposing bilayers and are represented as average interaction energy (in kBT units) per assembling SNAREpin (see Supplementary Methods). At low CPX concentration (0.3 μM), the separation profile shows two adhesions. The first one (at ~9 nm) corresponds to the unbinding of CPX-free SNAREpins that are ~70% assembled (down to approximately layer +6) and show a binding energy of 35 kBT (ref. 19). The second one (at ~16 nm) corresponds to the unbinding of CPX-bound SNAREpins that are ~50% assembled (down to approximately layer +1) and show a binding energy of 15 kBT (see text for details). () When more CPX is added between SNARE bilayers, the first adhesion (black) progressively decreases wh! ile the second one (purple) progressively increases (more SNAREpins are clamped by CPX). At high CPX concentration, all SNAREpins are clamped by CPX and show reduced binding energy and extent of assembly. * Figure 2: CPX allows SNAREpins to assemble at a larger distance. SNARE bilayers were approached down to a specific minimal interbilayer distance (dmin) and kept in contact for at least 30 min (which is the time necessary to reach an optimal adhesion19) before being separated (squares, approach; circles, separation). In the absence of CPX (red and black profiles), if dmin > ~9 nm, SNAREs do not assemble during the approach phase and no adhesion is observed upon separation; the red curve shows one example with dmin ~ 11 nm. In the presence of CPX (green profile), SNAREs form stable membrane-bridging complexes as soon as dmin < ~15 nm; the green curve gives one example where dmin ~ 14 nm. CPX thus allows the SNAREs to find each other and to assemble at a larger distance. * Figure 3: CPX activates and clamps SNAREpin assembly. Experiments similar to those presented in Figure 2 were repeated with many different dmin in the presence of various CPX variants (each data point corresponds to a single approach-separation cycle). Such experiments allow one to determine exactly at which distance SNAREpins start assembling and to measure the energy of assembly at a given distance. All data points were obtained with 1 μM of CPX, because at this concentration, >80% of the SNAREpins are bound to CPX (affinity better than 0.2 μM; see Fig. 4). In the absence of CPX, SNAREs do not begin to assemble until the bilayers are within ~9 nm, and they zipper into highly energetic (~35 kBT) SNAREpins. CPX allows the SNAREpins to zipper into an intermediate (~15 kBT) energetic state when the bilayers are as far as ~15 nm apart, suggesting an activation of SNAREpin assembly by CPX. The energy of this intermediate state is unchanged over a wide range of separations (dmin ~5–15 nm), suggesting a clamping effect of CPX on ! SNAREpin assembly. Activation of SNAREpin assembly is observed with all CPX variants tested and is thus controlled by the central helix of CPX. When the interbilayer distance becomes smaller than 9 nm, only mutants having the native or strengthened accessory helix can hold the intermediate (~15 kBT) state in place. Mutants with a deleted or weakened accessory helix cannot prevent C-terminal assembly of SNAREs, which leads to the highly energetic (~35 kBT) SNAREpin state19. * Figure 4: Affinity of CPX variants for the SNAREpin. The dissociation constants K between the SNAREpin and the wild-type CPX (purple), the nonclamping CPX(K26A) mutant (green) or the superclamping CPX(Q37A R41F Y44A Q48L) mutant (red) were estimated from the fraction of clamped SNAREpins (which is directly related to e2, the adhesion due to CPX-bound SNAREpins in Fig. 1d) at various CPX concentrations. For each mutant, at least three different concentrations were tested and the plot 1/e2 versus 1/[CPX] was fitted by a straight line whose slope gives the dissociation constant K (Supplementary Discussion and Supplementary Table 1). In the case of the superclamping mutant, only the two highest concentrations are showed here for clarity (the fit was, however, deduced from three different protein concentrations). The nonclamping CPX mutant shows a slightly lower affinity for SNAREpins than wild-type CPX, whereas the superclamping CPX mutant shows a much higher affinity for SNAREpins. * Figure 5: CPX reshapes the energy landscape of SNAREpin folding. CPX digs an adhesion well in the pathway of cognate SNARE assembly. In the absence of CPX (black), v- and t-SNAREs need to overcome a nonspecific repulsion barrier (ending at an interbilayer distance d ~ 9 nm) to begin their assembly and form highly energetic (~35 kBT) SNAREpins. In the presence of CPX (purple), an intermediate energetic state of lower energy (~15 kBT) appears on the folding pathway of SNAREpin. The nonspecific repulsion barrier still exists but is displaced further away, thus allowing v- and t-SNAREs to bind at a larger distance (~15 nm). This barrier at large distance is generated by the central helix of CPX, which facilitates N-terminal assembly of SNAREpins most likely by increasing the exposure of t-SNAREs (see text and Fig. 6 for details). It is followed by a second repulsion barrier at short distance generated by the accessory helix of CPX, which prevents further SNAREpin assembly by competing with the v-SNARE for C-terminal binding to the t-SNARE. Th! e dashed lines indicate the regions that are not observed in the SFA. Because the intermediate state is stable over an hour (typical duration of the contact time between two SNARE bilayers in the SFA), the energy difference between the binding energy and the barrier height (that is, the activation energy) must be higher than 30 kBT (see Supplementary Discussion). * Figure 6: CPX directly interacts with membrane-anchored t-SNAREs. (,) Interaction energy versus distance profile between two t-SNARE () or between two v-SNARE () bilayers in the absence (black) or the presence (purple) of 1 μM CPX (squares, approach; circles, separation). No adhesion is observed in both cases, either with or without CPX (during the separation phase, the interaction force continuously decreases until reaching the zero baseline). CPX affects the interaction profile of t-SNARE but not v-SNARE bilayers. In the presence of CPX, the long-range repulsive forces between t-SNARE bilayers begin 10 nm further away (the t-SNAREs see each other at a larger distance), and the distance at contact is about 1 nm larger (the protein layer between the two compressed bilayers is now thicker), suggesting that CPX binds to t-SNARE and increases its exposure toward solution (makes it more erected on the bilayer surface). Note that the repulsion profile between two v-SNARE bilayers is stronger than that between two t-SNARE bilayers, which is con! sistent with the fact that v-SNARE is largely unstructured. The insets show the approaching phase of the interaction profiles on a semi-log scale to more clearly display the repulsive forces. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Cell Biology, School of Medicine, Yale University, New Haven, Connecticut, USA. * Feng Li, * Frédéric Pincet, * Claudio G Giraudo, * David Tareste & * James E Rothman * Laboratoire de Physique Statistique, Unité Mixte de Recherche 8550, Centre National de la Recherche Scientifique associée aux Universités Paris VI et Paris VII, Ecole Normale Supérieure, Paris, France. * Feng Li, * Frédéric Pincet & * Eric Perez * Institut National de la Santé et de la Recherche Médicale, Unité 950, Paris, France. * David Tareste Contributions F.L. and C.G.G. made constructs and did protein purification. F.L. carried out SFA and ITC measurements. C.G.G. did cell-cell fusion assay. F.L., F.P. and D.T. analyzed the data. F.L., F.P., E.P., D.T. and J.E.R. interpreted the results and prepared the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * James E Rothman Author Details * Feng Li Search for this author in: * NPG journals * PubMed * Google Scholar * Frédéric Pincet Search for this author in: * NPG journals * PubMed * Google Scholar * Eric Perez Search for this author in: * NPG journals * PubMed * Google Scholar * Claudio G Giraudo Search for this author in: * NPG journals * PubMed * Google Scholar * David Tareste Search for this author in: * NPG journals * PubMed * Google Scholar * James E Rothman Contact James E Rothman Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–5, Supplementary Table 1, Supplementary Discussion and Supplementary Methods Additional data Entities in this article * Vesicle-associated membrane protein 2 Vamp2 Mus musculus * View in UniProt * View in Entrez Gene * Synaptosomal-associated protein 25 Snap25 Mus musculus * View in UniProt * View in Entrez Gene * Syntaxin-1A Stx1a Rattus norvegicus * View in UniProt * View in Entrez Gene * Complexin-1 Cplx1 Rattus norvegicus * View in UniProt * View in Entrez Gene * Complexin-3 CPLX3 Homo sapiens * View in UniProt * View in Entrez Gene - A RING E3–substrate complex poised for ubiquitin-like protein transfer: structural insights into cullin-RING ligases
- Nat Struct Mol Biol 18(8):947-949 (2011)
Nature Structural & Molecular Biology | Brief Communication A RING E3–substrate complex poised for ubiquitin-like protein transfer: structural insights into cullin-RING ligases * Matthew F Calabrese1, 4 * Daniel C Scott1, 2, 4 * David M Duda1, 2 * Christy R R Grace1 * Igor Kurinov3 * Richard W Kriwacki1 * Brenda A Schulman1, 2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:947–949Year published:(2011)DOI:doi:10.1038/nsmb.2086Received14 January 2011Accepted17 May 2011Published online17 July 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg How RING E3 ligases mediate E2-to-substrate ubiquitin-like protein (UBL) transfer remains unknown. Here we address how the RING E3 RBX1 positions NEDD8's E2 (UBC12) and substrate (CUL1). We find that existing structures are incompatible with CUL1 NEDD8ylation and report a new conformation of RBX1 that places UBC12 adjacent to CUL1. We propose RING domain rotation as a general mechanism for UBL transfer for the largest family of E3s. View full text Figures at a glance * Figure 1: UBC12–RBX1 interactions. () CUL1 NEDD8ylation mediated by WT and mutant UBC12in vitro. Mutated residues are orange on UBC12 (otherwise cyan)–RBX1 (pink) model. () 2D HSQC [15N-1H] spectra of [15N]UBC12core in the absence (red) and presence (blue) of equimolar RBX1RING. A subset of shifted resonances are labeled in the spectra. Residues with chemical shift differences >1 s.d. above the mean are labeled and colored orange on UBC12 (otherwise cyan)–RBX1 (pink) model. * Figure 2: Models from prior structures reveal an E2-to-substrate gap. () Structural model of CUL1CTD–RBX1–UBC12core (colored light green, pink, cyan, respectively) complex based on previous CUL1–RBX1 and RING–E2 structures2, 3, 4, 7, 8 (Supplementary Fig. 1). CUL1NEDD8 modification site and UBC12 catalytic cysteine are indicated by blue and yellow spheres, respectively (next to double arrow). () In vitroCUL1 NEDD8ylation assays using disulfide-linked (S~S) and unlinked (SH SH) "split'n'coexpress" CUL1–RBX1 (ref. 7). +DTT, with dithiothreitol. Pairs A–C refer to three distinct combinations of engineered cysteines (Supplementary Fig. 2). CUL1-NTD, CUL1 N terminus. * Figure 3: Structure of CUL1CTD–RBX1 in new conformation. () One copy of CUL1CTD (dark green)–RBX1 (purple) from the asymmetric unit. () Same as , but rotated ~70° in y and ~20° in x dimension. () Structural overlay with previous CUL1CTD (light green) oriented similarly to 7, 8. () Structural overlay with previous RBX1 (pink) in same orientation as . () Structural model of CUL1CTD–RBX1–UBC12core (light green, purple, cyan, respectively) complex based on the new RBX1 conformation. () Bis-maleimidoethane (BMOE) cross-linking between engineered cysteine mutants of Ubc12 and CUL1CTD. Residues mutated to cysteine are numbered on the model (left, view rotated by ~70° and ~40° about the x and z axes relative to ), and products of cross-linking reactions are shown for all possible Ubc12 + CUL1–RBX1 combinations (right). Note residue numbering for yeast Ubc12 (Sc. Ubc12). For reference, UBC12 catalytic cysteine and CUL1 NEDD8ylation site are shown as sticks. () In vitroCUL1 NEDD8ylation for UBC12 mutants at the predicted interfa! ce. Mutated residues are shown on the model (left). Accession codes * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3RTR * 3RTR Author information * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Matthew F Calabrese & * Daniel C Scott Affiliations * Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. * Matthew F Calabrese, * Daniel C Scott, * David M Duda, * Christy R R Grace, * Richard W Kriwacki & * Brenda A Schulman * Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. * Daniel C Scott, * David M Duda & * Brenda A Schulman * Cornell University, Department of Chemistry and Chemical Biology, Northeastern Collaborative Access Team (NE-CAT), Advanced Photon Source (APS), Argonne, Illinois, USA. * Igor Kurinov Contributions M.F.C. designed, performed and analyzed experiments, and wrote the manuscript. D.C.S. designed, performed and analyzed experiments. D.M.D. designed and analyzed experiments. C.R.R.G. designed and performed experiments. R.W.K. designed and analyzed experiments. I.K. designed and performed experiments. B.A.S. advised and assisted on all aspects of the project and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Matthew F Calabrese or * Brenda A Schulman Author Details * Matthew F Calabrese Contact Matthew F Calabrese Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel C Scott Search for this author in: * NPG journals * PubMed * Google Scholar * David M Duda Search for this author in: * NPG journals * PubMed * Google Scholar * Christy R R Grace Search for this author in: * NPG journals * PubMed * Google Scholar * Igor Kurinov Search for this author in: * NPG journals * PubMed * Google Scholar * Richard W Kriwacki Search for this author in: * NPG journals * PubMed * Google Scholar * Brenda A Schulman Contact Brenda A Schulman Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (14M) Supplementary Methods, Supplementary Figures 1–6, and Supplementary Tables 1 and 2 Additional data Entities in this article * Cullin-associated NEDD8-dissociated protein 1 CAND1 Homo sapiens * View in UniProt * View in Entrez Gene * Cullin-5 CUL5 Homo sapiens * View in UniProt * View in Entrez Gene * RING-box protein HRT1 HRT1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * E3 ubiquitin-protein ligase RBX1 RBX1 Homo sapiens * View in UniProt * View in Entrez Gene * Ubiquitin-conjugating enzyme E2 D1 UBE2D1 Homo sapiens * View in UniProt * View in Entrez Gene * DCN1-like protein 1 DCUN1D1 Homo sapiens * View in UniProt * View in Entrez Gene * NEDD8-conjugating enzyme UBC12 UBC12 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Ubiquitin-conjugating enzyme E2 R1 CDC34 Homo sapiens * View in UniProt * View in Entrez Gene * NEDD8-conjugating enzyme Ubc12 UBE2M Homo sapiens * View in UniProt * View in Entrez Gene * NEDD8 NEDD8 Homo sapiens * View in UniProt * View in Entrez Gene * S-phase kinase-associated protein 1 SKP1 Homo sapiens * View in UniProt * View in Entrez Gene * Cullin-1 CUL1 Homo sapiens * View in UniProt * View in Entrez Gene - DNA secondary structures and epigenetic determinants of cancer genome evolution
- Nat Struct Mol Biol 18(8):950-955 (2011)
Nature Structural & Molecular Biology | Analysis DNA secondary structures and epigenetic determinants of cancer genome evolution * Subhajyoti De1, 2 * Franziska Michor1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:950–955Year published:(2011)DOI:doi:10.1038/nsmb.2089Received06 December 2010Accepted04 May 2011Published online03 July 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 An unstable genome is a hallmark of many cancers. It is unclear, however, whether some mutagenic features driving somatic alterations in cancer are encoded in the genome sequence and whether they can operate in a tissue-specific manner. We performed a genome-wide analysis of 663,446 DNA breakpoints associated with somatic copy-number alterations (SCNAs) from 2,792 cancer samples classified into 26 cancer types. Many SCNA breakpoints are spatially clustered in cancer genomes. We observed a significant enrichment for G-quadruplex sequences (G4s) in the vicinity of SCNA breakpoints and established that SCNAs show a strand bias consistent with G4-mediated structural alterations. Notably, abnormal hypomethylation near G4s-rich regions is a common signature for many SCNA breakpoint hotspots. We propose a mechanistic hypothesis that abnormal hypomethylation in genomic regions enriched for G4s acts as a mutagenic factor driving tissue-specific mutational landscapes in cancer. View full text Figures at a glance * Figure 1: Spatial distribution of breakpoint hotspots in cancer genomes and genomes of healthy human subjects. () SCNA breakpoints can occur at high frequencies tens of kilobases away from EGFR (a known cancer gene shared across multiple cancer subtypes), shown in red. The direction of the red arrow shows the direction of transcription of EGFR. () SCNA breakpoints can occur at high frequency tens of kilobases away from PAX5 (a known cancer gene specific to acute lymphoblastic leukemia), shown in red. The red arrow shows the direction of transcription of PAX5. () SCNA breakpoint densities calculated over 1-Mb nonoverlapping genomic blocks across the human genome. Dotted vertical lines mark centromeres. () Summary statistics for SCNA breakpoint hotspots. Frequencies are shown in parentheses. * Figure 2: Association between G-quadruplex–forming sequences and breakpoint hotspots. () The distribution of the density (bp) of PG4s in 10-kb genomic blocks that have at least one SCNA breakpoint in cancer is markedly higher than the distribution of PG4s in those genomic blocks that harbor no breakpoints. The whiskers of the box plots represent the range of the PG4s density for the respective groups.() A schematic representation of DNA replication near a G4 structure and generation of an SCNA. Arrows indicate the direction of motion of the DNA polymerase. Only the leading strand obstructs the motion of the DNA polymerase and therefore SCNAs are more likely to occur at the 5′ side of G4 structures. () Cancer SCNAs with at least two PG4s within 10 kb are significantly likely to occur at the 5′ side of the G4 structures, an observation that is consistent with the hypothesis that these structures inhibit the action of DNA polymerase. Frequencies are shown within parentheses. The pattern is independent of the choice of parameters (see Supplementary Table 5). * Figure 3: Role of G-quadruplex structures in the generation of breakpoint hotspots. () Extent of differential methylation in colon cancer relative to normal colon (red), density of G4 sequences (orange) and density of DNA breakpoints in cancer (gray) are shown across the human chromosomes. Vertical dotted lines mark centromeres. A negative value of differential methylation indicates differential hypomethylation. () The density of DNA breakpoints in cancer is higher in genomic blocks that have both above-average hypomethylation and above-average PG4s density than that in genomic blocks that do not have above-average representation of either of the factors. The purple horizontal dashed line shows the median breakpoint density corresponding to the rightmost group. The whiskers of the box plots represent the range of the breakpoint frequencies for the respective groups. () SCNA breakpoint hotspots with above-average PG4s density are significantly differentially hypomethylated (low differential methylation score) relative to the genome-wide background. SCNA brea! kpoint hotspots specific to colorectal cancers with above-average PG4s density show a similar trend (P value > 0.05 because there are fewer data points). * Figure 4: A mechanistic hypothesis of epigenetic involvement in the generation of breakpoints in cancer genomes. Genomes in normal tissue are generally hypermethylated and stable. Genome-wide hypomethylation, which occurs stochastically during aging and tumorigenesis, offers a favorable environment in which PG4s can fold into G4 structures in the presence of stabilizing proteins and negative supercoiling. G4 structures are mutagenic and have the potential to generate deletion, insertion or rearrangement events of genetic material on which selection can act to drive cancer evolution. See Discussion for further details. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. * Subhajyoti De & * Franziska Michor * Department of Biostatistics, Harvard School of Public Health, Boston, Massachusetts, USA. * Subhajyoti De & * Franziska Michor Contributions S.D. and F.M. designed the research and wrote the manuscript. S.D. performed the research. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Franziska Michor Author Details * Subhajyoti De Search for this author in: * NPG journals * PubMed * Google Scholar * Franziska Michor Contact Franziska Michor Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (524K) Supplementary Figures 1–3, Supplementary Tables 1–9 and Supplementary Methods Additional data Entities in this article * Epidermal growth factor receptor EGFR Homo sapiens * View in UniProt * View in Entrez Gene * Paired box protein Pax-5 PAX5 Homo sapiens * View in UniProt * View in Entrez Gene - Transcription initiation platforms and GTF recruitment at tissue-specific enhancers and promoters
- Nat Struct Mol Biol 18(8):956-963 (2011)
Nature Structural & Molecular Biology | Resource Transcription initiation platforms and GTF recruitment at tissue-specific enhancers and promoters * Frederic Koch1, 2, 3, 10 * Romain Fenouil1, 2, 3, 10 * Marta Gut4, 5, 6, 10 * Pierre Cauchy1, 2, 3, 7 * Thomas K Albert8 * Joaquin Zacarias-Cabeza1, 2, 3 * Salvatore Spicuglia1, 2, 3 * Albane Lamy de la Chapelle1, 2, 3 * Martin Heidemann9 * Corinna Hintermair9 * Dirk Eick9 * Ivo Gut4, 6 * Pierre Ferrier1, 2, 3 * Jean-Christophe Andrau1, 2, 3 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:956–963Year published:(2011)DOI:doi:10.1038/nsmb.2085Received30 June 2010Accepted12 May 2011Published online17 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 Recent work has shown that RNA polymerase (Pol) II can be recruited to and transcribe distal regulatory regions. Here we analyzed transcription initiation and elongation through genome-wide localization of Pol II, general transcription factors (GTFs) and active chromatin in developing T cells. We show that Pol II and GTFs are recruited to known T cell–specific enhancers. We extend this observation to many new putative enhancers, a majority of which can be transcribed with or without polyadenylation. Importantly, we also identify genomic features called transcriptional initiation platforms (TIPs) that are characterized by large areas of Pol II and GTF recruitment at promoters, intergenic and intragenic regions. TIPs show variable widths (0.4–10 kb) and correlate with high CpG content and increased tissue specificity at promoters. Finally, we also report differential recruitment of TFIID and other GTFs at promoters and enhancers. Overall, we propose that TIPs represent imp! ortant new regulatory hallmarks of the genome. View full text Figures at a glance * Figure 1: Pol II and GTF recruitment to T-cell stage-specific enhancers of active loci or genes poised for activation. (–) ChIP-seq binding profiles for GTFs, CBP, total, initiating (Ser5P) and elongating (Ser2P) Pol II, active chromatin marks and FAIRE (accessible DNA regions). Light gray vertical bands show previously annotated or characterized enhancer regions, and dark gray bands indicate promoters. Normalized ChIP-seq signals for each experiment are shown on the right. Conservation, mappability track, regulatory elements and genes on positive (+) or negative (−) strand are indicated below the ChIP-seq lanes. In and at the active Cd4 and Cd8 loci, GTFs and initiating (Ser5P) Pol II are detected at proximal enhancer (PE) and thymocyte enhancer (TE), as opposed to the distal enhancer (DE), elements (Cd4) and EI, EII and EV (Cd8). At the Il2ra inactive locus (), poised for transcription and activated either before or after the double-positive differentiation stage, Ser5P and GTFs are detected at a previously characterized enhancer (element V) region. The binding profiles of the remainin! g GTFs are shown in Supplementary Figure 2a–c. * Figure 2: Epigenetic or transcriptional features and tissue-specific expression of putative enhancers recruiting Ser5P and TBP. () Average binding profiles of GTFs, Pol II, active chromatin marks, CBP and FAIRE in IGRs (centered on the maximum TBP signal) and non-oriented promoters (centered on the TSS; see Supplementary Fig. 3d for oriented genes). () Tissue-specific expression of genes associated with promoters or adjacent to putative enhancer IGRs. Using microarray data, associated genes were analyzed for their expression in various tissues and ordered by decreasing ratio with the whole genome (tissues with the highest five ratios are shown; see Supplementary Fig. 4 for all tissues). () Genes associated with TBP and Ser5P IGRs show a more tissue-restricted expression compared to promoters and to other selections. Box plot of expression for IGRs and promoters in double-positive cells and the remaining tissues are shown. The differential (red and blue bars) is greater for IGRs (left). The same analysis was conducted using different IGR-selection criteria including H3K4me1 and H3K4me3 (yellow), CBP a! nd H3K4me1 (orange) and CBP-H3K4me1-H3K4me3 (green). Our TBP-Ser5P-H3K4me1-H3K4me3 (blue) selection showed the highest tissue-restricted expression as well as the highest expression levels of IGR-associated genes (right). Similar analyses using all hematopoietic tissues are shown in Supplementary Figure 4c. () Validation of enhancer activity of two TBP and Ser5P IGRs in a promoter-dependent luciferase reporter assay. The Dusp6 and Rhoh promoters and IGRs were cloned in a pGL3 vector and transfected in a T-cell line (EL4). The Dusp6 IGR was cloned into both orientations and retained its ability to enhance promoter-driven expression. Error bars represent s.e.m. from two independent transfections. The complete experiment is presented in Supplementary Figure 11c. * Figure 3: TBP and Ser5P enhancers are transcribed with or without polyadenylation. () Examples of known enhancers transcribed in the presence (E8I element of the Cd8 locus, left) or absence (PE element of the Cd4 locus, right) of polyadenylation signal. Total and poly(A) RNAs signals are represented below the TBP and Ser5P ChIP-seq lanes as log2 signals of the directional RNA-seq experiments. RNA strand orientation is indicated on the left. Transcribed enhancer elements are indicated by a light gray vertical bands. Additional examples of IGR transcription are shown in Supplementary Figure 7. Possible tracking of Pol II toward Cd8a is indicated by the dotted arrow below the Ser5P lane. () Pol II ChIP-seq and oriented RNA-seq average profiling on TBP and Ser5P enhancer IGRs or gene promoters (left panels) for total (top panels) or poly(A) RNAs (bottom panels). Selected TBP and Ser5P IGRs were divided into three populations associated with either poly(A), no poly(A) or no RNA, and orientation of the IGRs was established based on the RNA levels. Signals are ce! ntered on the TSS of genes or on the main TBP peak of IGRs. * Figure 4: Poly(A) and non-poly(A) IGR subpopulations show distinct chromatin signatures between each other and genes. Comparison of active chromatin marks, CBP and ETS1 on oriented IGRs and promoters. ChIP-seq average binding profiles of the IGR populations described in Figure 3b are shown for genes and poly(A) (upper panels), no poly(A) and no RNA IGRs (lower panels). The profiles for the remaining factors described in this study are shown in Supplementary Figure 7d. * Figure 5: Pol II and GTFs transcription initiation platforms. () Examples of transcriptional initiation (TBP, Ser5P) or elongation (Ser2P, H3K36me3) hallmarks on TIPs at promoters, intragenic or IGR locations from left to right, respectively. The TBP and Ser5P TIPs isolated using a systematic approach (see Online Methods) are indicated by a red horizontal bar below TBP and Ser5P ChIP-seq signals. () Heatmaps of TIPs sorted by size and anchored on their center. TIPs (based on TBP and Ser5P selection) are shown for TBP, Ser5P and for the corresponding profiles for GTFs, active chromatin marks, FAIRE and the RNA-seq signal for positive and negative strands. TIP boundaries are represented by a red 5′ (right side) and green (left side) 3′ line. Heatmaps for all ChIP-seq and RNA-seq experiments at promoters, intragenic and IGR locations (as well as for input or mock Ig controls) are included in Supplementary Figure 9b,c. * Figure 6: TIPs correlate with CpG content and tissue-specific expression at promoters. () CpG content across TIPs anchored on their center, similarly to Figure 5b. Promoters clearly show the highest CpG content. Similar trends are also visible in IGRs and, to a lesser extent, in intragenic regions. () Clustering of T-cell and non–T-cell transcription factor motifs at TIPs around promoters. Most putative TFBS overlap with the high CpG content from . () Analysis of tissue specificity of expression for genes associated to promoter TIPs, similarly to Figure 2b. The associated genes show a more pronounced double-positive T-cell gene-expression pattern, indicating an increased tissue specificity at TIPs. The remaining genomic regions are analyzed in Supplementary Figure 10a. () Genes associated with promoter TIPs were classed into four equally sized groups (quartiles) with increasing platform size. The tissue specificity of the expression pattern increases with platform size, as indicated by the increasing rank and decreasing associated P values. The complete rank! s are presented in Supplementary Figure 10b. () Correlation of TIP size to absolute gene expression levels at promoters (r = 0.33). The global and local fitted curves are represented by solid red and dashed black lines, repectively. Similar graphs for IGRs and intragenic regions are shown in Supplementary Figure 10c. TIP size and expression values were transformed using a hyperbolic arcsine (Asinh) function. * Figure 7: Average profiles of TIPs and model summarizing their features at distinct genomic locations. () Average profiles of TBP, Ser5P, TFIIB, TFIIH, TFIIE, active chromatin marks, ETS1 and FAIRE across all resized TIPs. Regions were divided into promoter (red), IGR (blue) and intragenic (green) locations (see Supplementary Fig. 12a for total profiles). In general, Ser5P, GTFs, ETS1, FAIRE and H3K4me3 are largely enriched throughout the platforms, H3K4me1 peaks just after the boundaries and H3K36me3 is depleted. IGR and intragenic profiles show mostly similar patterns, including lower TBP, Ser5P and H3K4me3 as well as higher TFIIH, TFIIE, H3K4me1 and ETS1 levels, compared to promoters. The remaining profiles are shown in Supplementary Figure 12b. () Total RNA signal across the different classes of TIPs. RNAs from the positive (blue) and negative (red) strands are shown. Transcription appears to start at either border, increases toward the opposite boundary and decreases again afterwards, indicative of a transcriptional barrier possibly imposed by H3K4me1. () Promoter, IGRs ! and intragenic TIPs are all characterized by open chromatin regions and are delimited by an enrichment of the H3K4me1 (Me1, green circles) histone mark (to a lesser extent at promoter), possibly reflecting a nucleosomal barrier. These areas have transcription initiation hallmarks such as Ser5P Pol II (green), H3K4me3 (Me3, yellow circles)—though less pronounced in IGRs and intragenic regions—and GTF recruitment in common. They differ in their relative proportions of ETS1 (designated here as TF) and GTFs at IGRs, compared to promoters. Promoters fundamentally differ from other TIPs in their ability to allow Pol II to enter elongation (blue), although Ser2P is not detected in the immediate proximity of TIPs (most likely because of higher elongation rate and less Ser2P accumulation at the 5′ ends). CpG and TFBS are more prominent at promoters, although TFs, such as ETS1, are more often recruited to enhancers. Bidirectional transcription is present at both promoter37, 38 ! and IGRs (Fig. 3b and Supplementary Fig. 9d). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE29362 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Frederic Koch, * Romain Fenouil & * Marta Gut Affiliations * Centre d'Immunologie de Marseille-Luminy, Université Aix-Marseille, Campus de Luminy, Marseille, France. * Frederic Koch, * Romain Fenouil, * Pierre Cauchy, * Joaquin Zacarias-Cabeza, * Salvatore Spicuglia, * Albane Lamy de la Chapelle, * Pierre Ferrier & * Jean-Christophe Andrau * Centre National de la Recherche Scientifique, UMR6102, Marseille, France. * Frederic Koch, * Romain Fenouil, * Pierre Cauchy, * Joaquin Zacarias-Cabeza, * Salvatore Spicuglia, * Albane Lamy de la Chapelle, * Pierre Ferrier & * Jean-Christophe Andrau * Institut National de la Santé et de la Recherche Médicale, U631, Marseille, France. * Frederic Koch, * Romain Fenouil, * Pierre Cauchy, * Joaquin Zacarias-Cabeza, * Salvatore Spicuglia, * Albane Lamy de la Chapelle, * Pierre Ferrier & * Jean-Christophe Andrau * Centre National de Génotypage, Commissariat à l'Energie Atomique, Evry, France. * Marta Gut & * Ivo Gut * Fondation Jean Dausset—Centre d'Etude du Polymorphisme Humain, Paris, France. * Marta Gut * Centre Nacional D'Anàlisi Genòmica, Parc Científic de Barcelona, Baldiri i Reixac, Barcelona, Spain. * Marta Gut & * Ivo Gut * Techniques Avancées pour le Génome et la Clinique, Marseille, France. * Pierre Cauchy * Institute of Molecular Tumor Biology, Medical Faculty of the Westfälische Wilhelms-Universität, Münster, Germany. * Thomas K Albert * Department of Molecular Epigenetics, Helmholtz Center Munich, Center of Integrated Protein Science, Munich, Germany. * Martin Heidemann, * Corinna Hintermair & * Dirk Eick Contributions J.-C.A., F.K., T.K.A., P.F. and I.G. conceived the framework of the study. J.-C.A. and F.K. designed the experiments. R.F., P.C. and F.K. carried out the bioinformatic analyses and data treatment. D.E., C.H. and M.H. produced and provided the Ser2P and Ser5P antibodies as well as other antibodies that were not presented in this study. All ChIP-seq and RNA-seq materials were prepared by F.K. with the exception of ETS1 ChIP-seq, which was prepared by P.C., M.G. and I.G. conducted all ChIP-seq and RNA sequencing experiments. J.Z.-C. and S.S. did the FAIRE experiment. F.K. did the cloning and luciferase experiments and A.L.d.l.C. participated and provided technical assistance. J.-C.A. wrote the manuscript, and F.K., R.F. and P.C. participated in its preparation. All authors reviewed the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Jean-Christophe Andrau or * Ivo Gut or * Pierre Ferrier Author Details * Frederic Koch Search for this author in: * NPG journals * PubMed * Google Scholar * Romain Fenouil Search for this author in: * NPG journals * PubMed * Google Scholar * Marta Gut Search for this author in: * NPG journals * PubMed * Google Scholar * Pierre Cauchy Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas K Albert Search for this author in: * NPG journals * PubMed * Google Scholar * Joaquin Zacarias-Cabeza Search for this author in: * NPG journals * PubMed * Google Scholar * Salvatore Spicuglia Search for this author in: * NPG journals * PubMed * Google Scholar * Albane Lamy de la Chapelle Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Heidemann Search for this author in: * NPG journals * PubMed * Google Scholar * Corinna Hintermair Search for this author in: * NPG journals * PubMed * Google Scholar * Dirk Eick Search for this author in: * NPG journals * PubMed * Google Scholar * Ivo Gut Contact Ivo Gut 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 * Jean-Christophe Andrau Contact Jean-Christophe Andrau 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 (10M) Supplementary Figures 1–12, Supplementary Tables 1 and 2, and Supplementary Methods Additional data Entities in this article * DNA-binding protein Ikaros Ikzf1 Mus musculus * View in UniProt * View in Entrez Gene * DNA nucleotidylexotransferase Dntt Mus musculus * View in UniProt * View in Entrez Gene * T-cell receptor beta chain Tcrb Mus musculus * View in Entrez Gene * T-cell receptor alpha chain Tcra Mus musculus * View in Entrez Gene * T-cell surface glycoprotein CD4 Cd4 Mus musculus * View in UniProt * View in Entrez Gene * CREB-binding protein Crebbp Mus musculus * View in UniProt * View in Entrez Gene * Transcription initiation factor TFIID subunit 1 Taf1 Mus musculus * View in UniProt * View in Entrez Gene * TATA-box-binding protein Tbp Mus musculus * View in UniProt * View in Entrez Gene * V(D)J recombination-activating protein 2 Rag2 Mus musculus * View in UniProt * View in Entrez Gene * V(D)J recombination-activating protein 1 Rag1 Mus musculus * View in UniProt * View in Entrez Gene * T-cell surface glycoprotein CD3 delta chain Cd3d Mus musculus * View in UniProt * View in Entrez Gene * Transcription initiation factor IIB Gtf2b Mus musculus * View in UniProt * View in Entrez Gene * Protein C-ets-1 Ets1 Mus musculus * View in UniProt * View in Entrez Gene * Interleukin-2 receptor subunit alpha Il2ra Mus musculus * View in UniProt * View in Entrez Gene * T-cell surface glycoprotein CD8 beta chain Cd8b1 Mus musculus * View in UniProt * View in Entrez Gene * T-cell surface glycoprotein CD8 alpha chain Cd8a Mus musculus * View in UniProt * View in Entrez Gene * Rho-related GTP-binding protein RhoH Rhoh Mus musculus * View in UniProt * View in Entrez Gene * Dual specificity protein phosphatase 6 Dusp6 Mus musculus * View in UniProt * View in Entrez Gene
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