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• Closing the circle on ribonucleotide reductases
  - Nat Struct Mol Biol 18(3):251-253 (2011)
  Nature Structural & Molecular Biology | News and Views Closing the circle on ribonucleotide reductases * Derek T Logan1Journal name:Nature Structural & Molecular BiologyVolume: 18,Pages:251–253Year published:(2011)DOI:doi:10.1038/nsmb0311-251Published online04 March 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. In this issue, a wide array of structural and biochemical techniques are applied to reveal the molecular details of activity regulation in one of life's most essential enzymes, the ribonucleotide reductase. View full text Figures at a glance * Figure 1: The three different classes of RNR6, 7, 11, 20, 21. Models of the active class I α2β2 complex (based on the crystal structures of the R1 and R2 proteins, PDB 1RLR and 1RIB); class III (1HK8), dimeric class II (PDB 30O0) and monomeric class II (PDB 1LIL; C. Drennan, Massachusetts Institute of Technology, personal communication). The monomeric class II enzymes have maintained within one polypeptide chain the minimal structural elements required for substrate specificity regulation21. Substrates and allosteric effectors are shown as space-filling models. The adenosylcobalamin cofactors in the class II enzymes are shown as sticks. The box at the upper right shows a homology model of a β-monomer for the class III enzymes. * Figure 2: Schematic overview of the two modes of allosteric regulation in ribonucleotide reductases. The overall activity is governed by the binding of dATP (inhibition) or ATP (stimulation) to the activity site, located in a small N-terminal ATP cone domain of the α2 subunit of RNRs from class Ia and III. With very few exceptions, class II RNRs lack ATP cones and are not activity regulated. The substrate specificity is regulated by the binding of dNTPs to the specificity site at the dimer interface: ATP and dATP upregulate the reduction of CDP and UDP, dTTP upregulates GDP reduction and dGTP increases the rate of ADP reduction. Loop 2 is a flexible loop involved in transmission of the specificity signal. * Figure 3: Different architectures of inactive RNR complexes. In eukaryotes, the α6β2 complex seems to predominate3, 16, 22. In E. coli17 and Pseudomonas aeruginosa23 RNR (M. Crona and B.-M. Sjöberg, Stockholm University, and A. Hofer, Umeå University, personal communication), dATP probably does not induce an α4β4 complex in which all ATP cones are in contact, as this would exclude β2 binding. Instead, a more open complex in which only two ATP cones (represented by the blue shapes in the lower part of the figure) are in contact seems more likely. Such organization would also explain why α4 tetramers are not observed alone but only in complex with β2. Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Derek T. Logan is in the Department of Biochemistry and Structural Biology at Lund University, Lund, Sweden. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Derek T Logan Author Details * Derek T Logan Contact Derek T Logan Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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
• Glutamate receptor ion channels: where do all the calories go?
  - Nat Struct Mol Biol 18(3):253-254 (2011)
  Nature Structural & Molecular Biology | News and Views Glutamate receptor ion channels: where do all the calories go? * Mark L Mayer1Journal name:Nature Structural & Molecular BiologyVolume: 18,Pages:253–254Year published:(2011)DOI:doi:10.1038/nsmb0311-253Published online04 March 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. Glutamate receptor ion channels use the free energy of ligand binding to trigger ion channel activation and desensitization. In this issue, an analysis of all-atom molecular dynamics simulations dissects the binding process, reveals a substantial gain in free energy produced by domain closure for agonists and reports unique energy landscapes for individual ligands. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Mark L. Mayer is at the Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Mark L Mayer Author Details * Mark L Mayer Contact Mark L Mayer Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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(3):255 (2011)
  Nature Structural & Molecular Biology | Research Highlights Research highlights Journal name:Nature Structural & Molecular BiologyVolume: 18,Page:255Year published:(2011)DOI:doi:10.1038/nsmb0311-255Published online04 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. Grow old with me Proteostasis—the ability of a cell or organism to control the amounts, conformations and locations of proteins—is influenced by many cellular processes, including translation, protein folding and degradation. A reduction in proteostasis capacity has been associated with ageing. Hoppe and colleagues now reveal a relationship between ubiquitin-mediated proteolysis and regulation of longevity. Working with the nematode Caenorhabditis elegans, the authors found that combined mutation of two factors involved in the ubiquitin-proteasome system, the chaperone-like ATPase CDC-48 (also known as p97 in mammals) and the deubiquitinase ATX-3, resulted in a considerable increase in worm lifespan (25.5 days on average, compared to 17.5 days in the wild-type parental strain). The physical interaction between CDC-48/p97 and the human Machado-Joseph disease related ATX-3 was previously known, though its functional relevance was unclear. The increased longevity of the cdc-48 atx-3 mutant ! occurs via the insulin–IGF-1 signaling pathway and depends on the FOXO transcription factor DAF-16. A model substrate was found to be stabilized in the cdc-48 atx-3 mutant, with an increase in polyubiquitinated forms enriched in non-K48 linkages. However, the overall levels of ubiquitinated proteins were no different in the double mutant than in wild-type cells, indicating that only specific endogenous substrates are targeted. The authors propose that CDC-48 and ATX-3 work together to edit ubiquitin chains on specific substrates and coordinate the formation of chains that are appropriate for proteasomal degradation. In the cdc-48 atx-3 mutant, proteins involved in the insulin–IGF-1 signaling pathway might have abnormal ubiquitin chains and not be efficiently targeted to or degraded by the proteasome, resulting in longer lifespan; in support of this model, overexpression of CDC-48 (which could also result in abnormal ubiquitin chain editing) also increased longevity. The! authors excluded DAF-16 as a direct target of CDC-48 and ATX-! 3, as its levels and cellular localization were not affected in the double mutant. Uncovering the identities of the relevant substrates will provide further insight into longevity regulation and also open avenues for therapeutic interventions. (Nat. Cell Biol. doi:10.1038/ncb2200, published online 13 Feb 2011) IC Self-assembling cartwheels Centrioles are important for the assembly of cilia, flagella and centrosomes and are characterized by a ninefold symmetrical structure resembling a cartwheel, with a central ring-like hub from which nine spokes radiate outwards. This cartwheel structure is thought to act as a scaffold onto which centriolar microtubules assemble, and it is the subject of two recent studies. Reporting in Cell, the groups of Gönczy and Steinmetz show, using structural analyses, that the centriolar protein SAS-6 from the nematode Caenorhabditis elegans forms rod-shaped homodimers through a strong interaction between their central coiled-coil domains. Analytical ultracentrifugation reveals the formation of higher-order oligomers in solution, which is mediated by the interaction between the N-terminal globular domains from adjacent homodimers. Experiments with transgenic worms and human cells confirm the physiological significance of SAS-6 oligomerization for centriole formation. Additional X-ray! structures of the N-terminal domain and part of the coiled-coil domain of the related protein Bld12p from the green alga Chlamydomonas reinhardtii were used to generate a structural model in which nine homodimers assemble into a ring-like structure from which nine spokes corresponding to the coiled-coil domains emanate. In fact, recombinant Bld12p self-assembles into remarkably similar structures, as shown by EM. In Science, van Breugel et al. report the X-ray structure of the N-terminal domain of SAS-6 from zebrafish, which forms a head-to-head dimer. A crucial point mutation that prevents dimerization in vitro was validated in vivo, as the corresponding mutant failed to rescue aberrant flagellar formation in a Bld12p null mutant. The authors postulated that SAS-6 could also dimerize through its coiled-coil domain, a prediction that was confirmed through structural and genetic analysis. In fact, an SAS-6 mutant in which the coiled-coil domain and its packing against the N! -terminal head domain are disturbed is unable to rescue the nu! ll phenotype, indicating that both dimerization interfaces have functional significance. An SAS-6 construct containing the two interfaces forms higher-order oligomers in solution, and cryo-EM images show a ring-like architecture with radially projecting spokes. Among structural models of SAS-6 rings with different symmetries, a nine-fold symmetric ring matches the diameter of cartwheel hubs observed in procentrioles by cryo-EM. Combined, these findings provide a structural basis for centriole formation that is evolutionarily conserved. (Science doi:10.1126/science.1199325, published online 27 Jan 2011; Cell144, 364–375, 2011) AH View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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
• The prospects for designer single-stranded RNA-binding proteins
  - Nat Struct Mol Biol 18(3):256-261 (2011)
  Nature Structural & Molecular Biology | Perspective The prospects for designer single-stranded RNA-binding proteins * Joel P Mackay1 * Josep Font1 * David J Segal2, 3 * Affiliations * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:256–261Year published:(2011)DOI:doi:10.1038/nsmb.2005Published online27 February 2011 Abstract * Abstract * Accession codes * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Spectacular progress has been made in the design of proteins that recognize double-stranded DNA with a chosen specificity, to the point that designer DNA-binding proteins can be ordered commercially. This success raises the question of whether it will be possible to engineer libraries of proteins that can recognize RNA with tailored specificity. Given the recent explosion in the number and diversity of RNA species demonstrated to play roles in biology, designer RNA-binding proteins are set to become valuable tools, both in the research laboratory and potentially in the clinic. Here we discuss the prospects for the realization of this idea. View full text Figures at a glance * Figure 1: Possible uses of engineered RNA-binding proteins (RBPs). () Fusing the eukaryotic translation initiation factor eIF4G to an RBP targeted to the 5′ untranslated repeat (5′ UTR) of a messenger RNA (mRNA) could drive translation. () An RBP that binds near the translational start codon could inhibit translation of an mRNA. () A 5′ splice site–binding RBP could block recruitment of the U1 component of the spliceosome, favoring the skipping of that exon. () Conversely, an RBP that recognizes a splicing enhancer site and is fused to an arginine- and serine-rich (RS) domain could favor inclusion of the associated exon. () An RBP fused to a fluorescent protein (such as GFP) could be used to track RNA localization in living cells. () An RBP fused to a nuclear localization signal (NLS) could be used to alter RNA localization. () An RBP fused to a protein such as Argonaute 2 (Ago2) and targeted to the 3′ UTR of an mRNA could promote the degradation of the message. () Fusion of an RBP to a nonspecific RNase could allow the cutting of! a specific target RNA. This approach would work best using a split-RNase strategy analogous to that used for the successful zinc-finger nucleases, preventing widespread cleavage throughout the cell. () An RBP that tightly bound a specific noncoding RNA (ncRNA) could block its activity, providing a useful functional probe. * Figure 2: Structures of RNA-recognition motifs (RRMs) and pumilio repeat (PUF) domain complexes with single-stranded RNAs (ssRNAs). () Structure of the Fox-1 RRM (blue) bound to a seven-base RNA (gray)26 (PDB accession code 2ERR). () Overlay of the structures of SRp20–RNA (slate and gray; PDB accession code 2I2Y)57 and U2AF65–RNA (orange and light orange, PDB accession code 2G4B)58. Nucleotides that contact each protein are numbered, color coded and represented as sticks. The similarities and differences in RNA-recognition mode can be seen. () Structure of the PUM1 PUF domain bound to an ssRNA target29 (PDB accession code 3BSX). () Close-up of the PUM1–RNA complex, showing the interactions made by two repeats with their target bases. Hydrogen bonds are orange dashed lines, and the tyrosine that stacks between the two bases is also shown. Cys1007 makes van der Waals contacts with A5. () Structure of the tandem K homology (KH) domains from NusA bound to an 11-base RNA target40 (PDB accession code 2ASB). The orientations of the two KH domains are related by an approximately 90° rotation about the hor! izontal axis. () Close-up of the NusA–RNA complex, showing base-specific interactions made to three of the nucleotides. The color scheme is as in , with oxygen and nitrogen atoms colored red and blue, respectively. A45 makes contacts with both KH1 and KH2. * Figure 3: Structures of zinc-finger (ZF)–RNA complexes. () Structure of the tandem CCCH ZF domains of Tis11d bound to an adenine- and uridine-rich target RNA44 (PDB accession code 1RGO). () Structure of the double zinc knuckle from the HIV-1 nucleocapsid protein bound to the SL3 recognition element47 (PDB code 1A1T). () Structure of a ZF from ZRANB2 bound to a single-stranded GGU-containing oligonucleotide54 (PDB accession code 3G9Y). Accession codes * Abstract * Accession codes * Author information Referenced accessions Protein Data Bank * 2ERR * 2I2Y * 2G4B * 3BSX * 2ASB * 1RGO * 1A1T * 3G9Y * 2ERR * 2I2Y * 2G4B * 3BSX * 2ASB * 1RGO * 1A1T * 3G9Y Author information * Abstract * Accession codes * Author information Affiliations * School of Molecular Bioscience, University of Sydney, New South Wales, Sydney, Australia. * Joel P Mackay & * Josep Font * Department of Pharmacology, University of California at Davis, Davis, California, USA. * David J Segal * Genome Center, University of California at Davis, Davis, California, USA. * David J Segal Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Joel P Mackay or * David J Segal Author Details * Joel P Mackay Contact Joel P Mackay Search for this author in: * NPG journals * PubMed * Google Scholar * Josep Font Search for this author in: * NPG journals * PubMed * Google Scholar * David J Segal Contact David J Segal Search for this author in: * NPG journals * PubMed * Google Scholar Additional data
• Genetic selection designed to stabilize proteins uncovers a chaperone called Spy
  - Nat Struct Mol Biol 18(3):262-269 (2011)
  Nature Structural & Molecular Biology | Article Genetic selection designed to stabilize proteins uncovers a chaperone called Spy * Shu Quan1, 2 * Philipp Koldewey1, 2 * Tim Tapley1, 2 * Nadine Kirsch1, 2 * Karen M Ruane3 * Jennifer Pfizenmaier1, 2 * Rong Shi3 * Stephan Hofmann1, 2 * Linda Foit1, 2 * Guoping Ren1, 2 * Ursula Jakob2 * Zhaohui Xu4, 5 * Miroslaw Cygler3, 6 * James C A Bardwell1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:262–269Year published:(2011)DOI:doi:10.1038/nsmb.2016Received27 July 2010Accepted15 December 2010Published online13 February 2011 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 To optimize the in vivo folding of proteins, we linked protein stability to antibiotic resistance, thereby forcing bacteria to effectively fold and stabilize proteins. When we challenged Escherichia coli to stabilize a very unstable periplasmic protein, it massively overproduced a periplasmic protein called Spy, which increases the steady-state levels of a set of unstable protein mutants up to 700-fold. In vitro studies demonstrate that the Spy protein is an effective ATP-independent chaperone that suppresses protein aggregation and aids protein refolding. Our strategy opens up new routes for chaperone discovery and the custom tailoring of the in vivo folding environment. Spy forms thin, apparently flexible cradle-shaped dimers. The structure of Spy is unlike that of any previously solved chaperone, making it the prototypical member of a new class of small chaperones that facilitate protein refolding in the absence of energy cofactors. View full text Figures at a glance * Figure 1: A dual fusion selection for enhancing in vivo protein stability. () Unstable test proteins inserted into β-lactamase are degraded by cellular proteases, producing penicillin-sensitive (PenVS) strains. Improving the folding of the test proteins increases penicillin resistance (PenVR)3. () Insertion of unstable test proteins into DsbA renders the strains sensitive to cadmium (CdCl2S). Improving the folding of the test proteins increases the strains' resistance toward cadmium (CdCl2R). () Thermodynamic stability of Im7 variants3 (given as ΔΔG0 relative to wild-type Im7) correlates with the minimal inhibitory concentration (MIC) of cadmium for cells expressing the DsbA fusions to these variants. () Penicillin resistance of cells expressing the β-lactamase–Im7 fusions correlates with cadmium resistance when they also express the DsbA-Im7 fusion proteins with the same Im7 variants. Error bars indicate the s.d. of three independent measurements. * Figure 2: Overall experimental scheme. Fusion constructs that link the stability of the poorly folded Im7 protein to PenVR (bla∷Im7) and to CdCl2R (dsbA∷Im7) were introduced into the same E. coli strain. Following mutagenesis by EMS treatment, mutants that simultaneously enhanced both PenVR and CdCl2R were selected, and levels of Im7 in these strains were measured. The mutants massively increased levels of Im7 as well as a host protein called Spy. Genetic experiments showed that increased Spy levels are necessary and sufficient to increase Im7 levels. The Spy protein was examined for molecular chaperone activity in vitro and found to be highly effective as a chaperone in preventing protein aggregation and aiding refolding. Spy was then crystallized, its structure was solved and mutants were made based on its structure to explore the interaction of Spy with substrates. * Figure 3: Im7 and Spy are abundant in the periplasm of EMS strains. () The PenVR and CdCl2R mutants EMS4 and EMS9 and the parental SQ1306 strain were transformed with plasmids encoding wild-type Im7 (WT) or the destabilized variants L53A I54A, I22V and F84A3, 6. Periplasmic extracts were prepared and analyzed by SDS-PAGE. () Spy overexpression is sufficient to enhance Im7 levels in both wild-type and ΔbaeSR backgrounds to those seen in EMS4. Plasmid-encoded Im7 and its destabilized variants were expressed in SQ765 wild-type (WT) or ΔbaeSR backgrounds, with or without the coexpression of plasmid-encoded Spy (designated as Spy++). Aggregated or insoluble proteins were not expected to be extracted using our periplasmic extraction procedure and therefore was not detected in these experiments. * Figure 4: Spy has chaperone activity. () Spy suppresses protein aggregation as monitored by light scattering. Aggregate formation in the absence (0:1) and presence of increasing amounts of Spy (ratios given are Spy:substrate) was monitored for thermally (43 °C) or chemically denatured substrates. () Spy enhances protein refolding as assessed by recovery of enzymatic activity. Refolding was monitored in the absence (0:1) and presence of Spy for thermally (43 °C) or chemically denatured substrates. Plots show mean ± s.d. of three independent measurements. Ultracentrifugation and gel-filtration studies (Supplementary Fig. 6) indicate that Spy is dimeric in solution, so Spy concentrations are given as a dimer. * Figure 5: Spy protects DsbB, aldolase and alkaline phosphatase from tannic acid–induced activity loss. Spy concentrations are given as a dimer. Plots show mean ± s.d. for three independent measurements. () Enzymatic activity of E. coli DsbB (0.5 μM) incubated in 100 μM tannic acid in the absence or presence of increasing amounts of Spy (ratios given are Spy:substrate). () Enzymatic activity of rabbit muscle aldolase (0.5 μM) incubated in 16 μM tannic acid in the absence or presence of increasing amounts of Spy. () Enzymatic activity of E. coli alkaline phosphatase (AP) (1 μM) incubated in 500 μM tannic acid in the absence or presence of increasing amounts of Spy. () spy and baeSR deletion strains are tannin sensitive. * Figure 6: Crystal structure of the Spy dimer shown in three orientations rotated by 90° along the vertical axis. For ease of comparison, the orientations shown in panels , and are identical. () Ribbon drawing shows an all–α-helical structure. One subunit is colored light blue and the other is colored magenta. The N and C termini and the secondary structural elements of the molecule are labeled. () Surface properties of Spy, colored as: backbone atoms, white; polar and charged side chain atoms, green; hydrophobic side chain atoms, yellow. Two predominantly hydrophobic patches in the concave surface are indicated as P1 (composed of Leu34, Ile42, Met46 and Ile103) and P2 (composed of Pro56, Met64, Ile68, Met85, Met93 and Met97). The residues labeled with fluorescent probes (for experiments in Fig. 7) are circled with black dashed lines. () Stereoview showing the cluster of hydrophobic residues at the tip of the cradle marked as P1 in . These residues are well conserved among homologous sequences (Supplementary Fig. 5). () Structural flexibility of Spy dimer. The molecular surface repre! senting Spy backbone atoms is colored based on the average B-factors for each residue. Note that the rim lining the concave surface has higher B-factors, indicating greater structural flexibility; in particular, the N and C termini are highly mobile. * Figure 7: Spy binds the disordered model substrate protein casein and the in vivo substrate protein Im7-L53A I54A. () Analytical gel filtration of Spy, casein or a 1:1 mixture of Spy and casein. The molecular weight of a dimer of Spy is 31 kDa, but it elutes as a 44-kDa molecule, consistent with the elongated form of the dimer seen in the crystal structure. The molecular weight of casein is 23–26 kDa. () Competition assay between urea-denatured Im7-L53A I54A and MDH for Spy in MDH refolding. Refolding of chemically denatured MDH was monitored by recovery of enzymatic activity plotted as a fraction of the activity of native (that is, nondenatured) MDH. Note that the curves of MDH alone (open squares) and MDH + Im7-L53A I54A (closed squares) overlap precisely. () Competition assay between casein and MDH for Spy binding. Refolding of chemically denatured MDH was monitored as in . () Normalized fluorescence emission spectra of acrylodan-labeled Spy mutants H96C and K77C in the absence or presence of an equimolar amount of casein. Note that the fluorescence change upon casein addition for S! py K77-acrylodan is opposite that from Spy H96C-acrylodan. () Normalized fluorescence emission spectra of acrylodan-labeled Spy A128C in the absence or presence of an equimolar amount of urea-denatured Im7-L53A I54A. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3O39 * 3O39 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. * Shu Quan, * Philipp Koldewey, * Tim Tapley, * Nadine Kirsch, * Jennifer Pfizenmaier, * Stephan Hofmann, * Linda Foit, * Guoping Ren & * James C A Bardwell * Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA. * Shu Quan, * Philipp Koldewey, * Tim Tapley, * Nadine Kirsch, * Jennifer Pfizenmaier, * Stephan Hofmann, * Linda Foit, * Guoping Ren, * Ursula Jakob & * James C A Bardwell * Department of Biochemistry, McGill University, Montreal, Quebec, Canada. * Karen M Ruane, * Rong Shi & * Miroslaw Cygler * Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA. * Zhaohui Xu * Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, USA. * Zhaohui Xu * Biotechnology Research Institute, National Research Council, Montreal, Quebec, Canada. * Miroslaw Cygler Contributions J.C.A.B. designed the study and wrote the manuscript, with contributions from M.C. and S.Q. S.Q., P.K., T.T., N.K., K.M.R., R.S., J.P., S.H. and G.R. conducted the experiments and collected and analyzed the data. J.C.A.B., Z.X. and M.C. further analyzed the data. L.F. and U.J. provided technical support and conceptual advice. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * James C A Bardwell Author Details * Shu Quan Search for this author in: * NPG journals * PubMed * Google Scholar * Philipp Koldewey Search for this author in: * NPG journals * PubMed * Google Scholar * Tim Tapley Search for this author in: * NPG journals * PubMed * Google Scholar * Nadine Kirsch Search for this author in: * NPG journals * PubMed * Google Scholar * Karen M Ruane Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer Pfizenmaier Search for this author in: * NPG journals * PubMed * Google Scholar * Rong Shi Search for this author in: * NPG journals * PubMed * Google Scholar * Stephan Hofmann Search for this author in: * NPG journals * PubMed * Google Scholar * Linda Foit Search for this author in: * NPG journals * PubMed * Google Scholar * Guoping Ren Search for this author in: * NPG journals * PubMed * Google Scholar * Ursula Jakob Search for this author in: * NPG journals * PubMed * Google Scholar * Zhaohui Xu Search for this author in: * NPG journals * PubMed * Google Scholar * Miroslaw Cygler Search for this author in: * NPG journals * PubMed * Google Scholar * James C A Bardwell Contact James C A Bardwell 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 1–7, Supplementary Table 1 and Supplementary Methods Additional data
• Structural and biochemical studies of the 5′3′ exoribonuclease Xrn1
  - Nat Struct Mol Biol 18(3):270-276 (2011)
  Nature Structural & Molecular Biology | Article Structural and biochemical studies of the 5′→3′ exoribonuclease Xrn1 * Jeong Ho Chang1, 3 * Song Xiang1, 2, 3 * Kehui Xiang1 * James L Manley1 * Liang Tong1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:270–276Year published:(2011)DOI:doi:10.1038/nsmb.1984Received22 August 2010Accepted22 November 2010Published online06 February 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The 5′→3′ exoribonucleases (XRNs) have important functions in transcription, RNA metabolism and RNA interference. The structure of Rat1 (also known as Xrn2) showed that the two highly conserved regions of XRNs form a single, large domain that defines the active site of the enzyme. Xrn1 has a 510-residue segment after the conserved regions that is required for activity but is absent from Rat1/Xrn2. Here we report the crystal structures of Kluyveromyces lactis Xrn1 (residues 1–1,245, E178Q mutant), alone and in complex with a Mn2+ ion in the active site. The 510-residue segment contains four domains (D1–D4), located far from the active site. Our mutagenesis and biochemical studies show that their functional importance results from their ability to stabilize the conformation of the N-terminal segment of Xrn1. These domains might also constitute a platform that interacts with protein partners of Xrn1. View full text Figures at a glance * Figure 1: Domain organization of Xrn1. Schematic drawing of the domain organizations of K. lactis Xrn1, human Xrn1 and S. pombe Rat1/Xrn2. The various domains are labeled. * Figure 2: Structure of K. lactis Xrn1. () Schematic drawing of the structure of K. lactis Xrn1. The domains are colored as in Figure 1 and labeled. The Mn2+ ion in the active site is shown as a dark gray sphere. (). Structure of K. lactis Xrn1, viewed along the arrow indicated in . () Stereoview of overlay of K. lactis Xrn1 and S. pombe Rat1/Xrn2 structures26. For simplicity, the linker between CR1 and CR2 and domains D1–D4 of Xrn1 are not shown. The structure of Xrn1 is colored as in , and that of Rat1/Xrn2 is in gray. All the structure figures were produced with PyMOL (http://www.pymol.org) unless stated otherwise. * Figure 3: Unique structural features in Xrn1. () Close-up of the structure of domain D1, as well as its interactions with CR1. The two three-stranded β-sheets in the periphery of the structure are labeled with the circled numbers in red. () Close-up of the structure of domains D2 and D4, as well as the interactions between D4 and CR1. * Figure 4: Structure of the active site of K. lactis Xrn1. () Molecular surface of K. lactis Xrn1, colored by electrostatic potential. The active site region is indicated with the yellow star. Domain D3 and the linker between CR1 and CR2 are not shown. () Molecular surface of S. pombe Rat1–Rai1 complex26, viewed in the same orientation as . Panels and were produced with the program Grasp50. () Stereo drawing of the active site of K. lactis Xrn1. Side chains in the active site are shown as stick models and labeled. The bound Mn2+ ion is shown as a gray sphere. () Overlay of the structures of the seven conserved acidic residues in CR1 and the two Tyr residues in CR2 in the active sites of Xrn1 (in cyan for carbon atoms) and Rat1/Xrn2 (in gray). The liganding interactions of the Mn2+ ion are indicated with dashed lines. Residues in Xrn1 are labeled in black, and those in Rat1/Xrn2 labeled in gray. * Figure 5: Biochemical studies on the nuclease activity of K. lactis Xrn1. () Time course showing the degradation of the 240-nt RNA substrate (indicated with the arrowhead, with 5′-end monophosphate and labeled at the 3′-end) by recombinant K. lactis Xrn1 (residues 1–1,245). () RNase assays showing the effect of site-specific and deletion mutations on the activity of K. lactis Xrn1. All reactions contained 20 ng of enzyme, except for the four lanes at the right, which contained 50 ng of enzyme. () Nuclease assays showing the effect of C-terminal deletion mutations on the activity of K. lactis Xrn1 toward a 55-nt ssDNA substrate, labeled at the 5′-end. () Nuclease assays showing the effect of deleting the N-terminal four residues on the activity of K. lactis Xrn1 toward the 55-nt ssDNA substrate. * Figure 6: RNA binding activity of K. lactis Xrn1. Electrophoretic mobility shift assays for wild-type and various deletion mutants of Xrn1. The wild-type enzyme has high affinity for the 240-nt RNA, producing a large complex that did not migrate into the gel. * Figure 7: Conserved surface features in the structure of K. lactis Xrn1. () Molecular surface of K. lactis Xrn1 colored by sequence conservation among fungal and mammalian enzymes, analyzed by the program ConSurf51. The colors vary from dark purple for highly conserved residues to cyan for residues with little conservation. A weakly conserved surface patch is highlighted with the red oval. () Surface conservation of Xrn1, viewed after a 90° rotation around the horizontal axis from . Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3PIE * 3PIF * 3PIE * 3PIF Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Jeong Ho Chang & * Song Xiang Affiliations * Department of Biological Sciences, Columbia University, New York, New York, USA. * Jeong Ho Chang, * Song Xiang, * Kehui Xiang, * James L Manley & * Liang Tong * Present address: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, P.R. China. * Song Xiang Contributions J.H.C. and S.X. carried out protein expression, purification and crystallization experiments. J.H.C., S.X. and L.T. carried out crystallographic data collection, structure determination and refinement. J.H.C. carried out mutagenesis, nuclease assays, EMSA and thermal shift experiments. K.X. helped with the preparation of the nuclease substrate. J.L.M. designed experiments and analyzed data. All authors commented on the manuscript. L.T. designed experiments, analyzed data and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Liang Tong Author Details * Jeong Ho Chang Search for this author in: * NPG journals * PubMed * Google Scholar * Song Xiang Search for this author in: * NPG journals * PubMed * Google Scholar * Kehui Xiang Search for this author in: * NPG journals * PubMed * Google Scholar * James L Manley Search for this author in: * NPG journals * PubMed * Google Scholar * Liang Tong Contact Liang Tong 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 (3M) Supplementary Figures 1–9 Additional data
• Common architecture of the flagellar type III protein export apparatus and F- and V-type ATPases
  - Nat Struct Mol Biol 18(3):277-282 (2011)
  Nature Structural & Molecular Biology | Article Common architecture of the flagellar type III protein export apparatus and F- and V-type ATPases * Tatsuya Ibuki1 * Katsumi Imada1, 2 * Tohru Minamino1, 3 * Takayuki Kato1 * Tomoko Miyata1 * Keiichi Namba1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:277–282Year published:(2011)DOI:doi:10.1038/nsmb.1977Received15 July 2010Accepted17 November 2010Published online30 January 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The proteins that form the bacterial flagellum are translocated to its distal end through the central channel of the growing flagellum by the flagellar-specific protein export apparatus, a family of the type III protein secretion system. FliI and FliJ are soluble components of this apparatus. FliI is an ATPase that has extensive structural similarity to the α and β subunits of FoF1-ATP synthase. FliJ is essential for export, but its function remains obscure. Here we show that the structure of FliJ derived from Salmonella enterica serovar Typhimurium is remarkably similar to that of the two-stranded α-helical coiled-coil part of the γ subunit of FoF1-ATP synthase and that FliJ promotes the formation of FliI hexamer rings by binding to the center of the ring. These results suggest that the type III protein export system and F- and V-type ATPases share a similar mechanism and an evolutionary relationship. View full text Figures at a glance * Figure 1: Structure of FliJ. () Cα ribbon drawing of FliJ. The binding regions for FlgN, FliT, FliH and both FliT and FliH are highlighted in blue, green, magenta and yellow, respectively. (,) Evolutionarily conserved residues of FliJ. The figures were prepared by ConSurf (http://consurf.tau.ac.il/)36. Residues are colored in accordance with conservation among amino acid sequences of FliJ from 50 different bacterial species. () The back view of . * Figure 2: Structural similarity between FliJ and the γ subunit of F1-ATPase. (,) Cα ribbon drawing of FliJ () and the γ subunit of bovine mitochondria F1-ATPase (PDB ID code 1E79)26 (). The α1 and α2 helices and the α/β domain of the γ subunit are colored blue, red and green, respectively. (,) Close-up view of the conserved region between FliJ and the γ subunit. FliJ (green) is superimposed on the γ subunit (light blue). Residues that are highly conserved among FliJ homologs and γ subunits are shown in red. () The ɛ subunit (pink; PDB ID code 1E79)26 is displayed with FliJ and the γ subunit. () The amino acid sequence of FliJ with various information. Green characters denote α-helices. Residues that are highly conserved between FliJ and the γ subunit are shown in red. The FlgN-, FliT- and FliH-binding regions are shown by yellow, blue and purple bars below the sequence, respectively. Asterisks above the sequence represent residues that are highly conserved among FliJs from various species. Dots below the sequence denote residues that we! re unresolved in the electron density. * Figure 3: Effect of FliJ on the formation of FliI rings. Negatively stained electron micrographs of FliI and FliI-FliJ mixtures. (–) FliI and FliJ were mixed at molar ratios of 1:0 (), 6:1 () and 1:1 (). Scale bar, 50 nm. * Figure 4: Electron micrograph of the FliI–FliJ complex. Negatively stained electron micrograph of a mixture of FliI and streptavidin-labeled FliJ. Most of the rings are paired. Close up views of the ring pairs are shown on the right. The densities bridging the pair are discernible. Scale bars, 100 nm (left) and 10 nm (right). * Figure 5: Comparison of the FliI and FliJ-FliI ring complexes. (,) Averaged cryo-EM images of the FliI () and FliJ-FliI () ring complexes embedded in vitreous ice. Hexameric features are clear in both images, but the central hole of the FliJ-FliI ring is filled in with an off-axis density. Scale bars, 5 nm. * Figure 6: Structure-based sequence alignment of FliI and the α and β subunits of F1-ATPase and pulldown assay to identify the FliI region that interacts with FliJ. () Structure-based sequence alignment of FliI and the α and β subunits of F1-ATPase from bovine mitochondria (1BMFA and 1BMFB) and the thermophilic Bacillus PS3 (1SKYA and 1SKYB). The purple and cyan bars indicate α-helices and β-strands, respectively. The region that interacts with FliJ is shown by the green box. Residues that are conserved between FliI and any of the F1 subunits are highlighted in red. () Interactions of FliJ with FliI, its fragments and deletion mutants were examined by pulldown assay using GST affinity chromatography. The eluted fractions were analyzed by both CBB staining and immunoblotting with polyclonal anti-FliI antibodies. * Figure 7: Schematic diagram showing a plausible model of the FliH–FliI–FliJ complex and its relative position and orientation to the export gate. FoF1-ATP synthase, which is composed of the α3β3 ring, the γ and ɛ subunits as the central stalk, the b and δ subunits as the peripheral stalk and the a and c subunits as the transmembrane domain, is shown in the right panel as a reference. Labels C, A and N represent the C-terminal, ATPase and N-terminal domains of FliI, respectively. The flagellar basal body composed of the MS ring, C ring and rod is colored gray. CM, cytoplasmic membrane. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3AJW * 3AJW Referenced accessions Protein Data Bank * 1E79 * 1E79 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan. * Tatsuya Ibuki, * Katsumi Imada, * Tohru Minamino, * Takayuki Kato, * Tomoko Miyata & * Keiichi Namba * Department of Macromolecular Sciences, Graduate School of Sciences, Osaka University, Osaka, Japan. * Katsumi Imada * Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Kawaguchi, Saitama, Japan. * Tohru Minamino Contributions T.I. prepared samples and carried out crystallization and X-ray structure analysis. K.I. helped and supervised T.I. in X-ray crystallography. T. Minamino prepared protein expression constructs and carried out pulldown assays. T.K. helped and supervised T.I. in cryo-EM image analysis. T. Miyata collected cryo-EM images. K.N. supervised the whole project. T.I., K.I., T. Minamino and K.N. wrote the paper based on discussions with T.K. and T. Miyata. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Katsumi Imada or * Keiichi Namba Author Details * Tatsuya Ibuki Search for this author in: * NPG journals * PubMed * Google Scholar * Katsumi Imada Contact Katsumi Imada Search for this author in: * NPG journals * PubMed * Google Scholar * Tohru Minamino Search for this author in: * NPG journals * PubMed * Google Scholar * Takayuki Kato Search for this author in: * NPG journals * PubMed * Google Scholar * Tomoko Miyata Search for this author in: * NPG journals * PubMed * Google Scholar * Keiichi Namba Contact Keiichi Namba 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 (320K) Supplementary Figure 1 and Supplementary Table 1 Additional data
• The hidden energetics of ligand binding and activation in a glutamate receptor
  - Nat Struct Mol Biol 18(3):283-287 (2011)
  Nature Structural & Molecular Biology | Article The hidden energetics of ligand binding and activation in a glutamate receptor * Albert Y Lau1, 2 * Benoît Roux1 * Affiliations * ContributionsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:283–287Year published:(2011)DOI:doi:10.1038/nsmb.2010Received17 August 2010Accepted30 November 2010Published online13 February 2011 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 Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate most excitatory synaptic transmission in the central nervous system. The free energy of neurotransmitter binding to the ligand-binding domains (LBDs) of iGluRs is converted into useful work to drive receptor activation. We have computed the principal thermodynamic contributions from ligand docking and ligand-induced closure of LBDs for nine ligands of GluA2 using all-atom molecular dynamics free energy simulations. We have validated the results by comparison with experimentally measured apparent affinities to the isolated LBD. Features in the free energy landscapes that govern closure of LBDs are key determinants of binding free energies. An analysis of accessible LBD conformations transposed into the context of an intact GluA2 receptor revealed that the relative displacement of specific diagonal subunits in the tetrameric structure may be key to the action of partial agonists. View full text Figures at a glance * Figure 1: Ligands of GluA2. The full agonists (top) are glutamate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and (S)-2-amino-3-(3-carboxy-5-methylisoxazol-4-yl)propionic acid (ACPA). The partial agonists (middle) are kainate, (S)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isothiazolyl)propionic acid (thio-ATPA), and (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]-4-isoxazolyl)propionic acid (4-AHCP). The antagonists (bottom) are (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid (ATPO), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and 6,7-dinitroquinoxaline-2,3 dione (DNQX). Crystal structures of each of these ligands in complex with the GluA2 LBD were used for our molecular models3, 6, 9, 22, 23, 24. * Figure 2: LBD conformational distributions. () The free energy landscapes that govern LBD closure for the holo and apo proteins calculated from all-atom umbrella sampling molecular dynamics simulations with explicit solvent. Each contour line corresponds to 1 kcal mol−1, with the darker colors indicating more favorable conformations. The free energy minimum associated with the most closed conformation is for AMPA: (ξ1, ξ2) = (9.2 Å, 8.4 Å). The conformation used for the ligand-docking simulations is (14.4 Å, 13.7 Å). These locations are indicated by the dotted lines in each panel. () The two-dimensional order parameter (ξ1, ξ2) describing closure of the GluA2 LBD. Each distance (dashed line) is measured between the centers of mass of the residues whose atoms are shown as spheres. The crystal structure of the open, apo LBD (1FTO) is shown. * Figure 3: Comparison of calculated free energy contributions with experimentally measured effective ligand-binding affinities to the isolated GluA2 LBD. () Calculated . () Calculated ΔGclose. () Calculated . In each plot, the solid line, which has a slope of 1, indicates perfect agreement between the calculated and experimental values. The dashed lines are linear regression fits to the data, and their slopes and correlation coefficients are shown. Each ligand is marked numerically in increasing order from the highest experimentally measured affinity to the lowest (Supplementary Table 1): 1, AMPA; 2, ACPA; 3, 4-AHCP; 4, CNQX; 5, glutamate; 6, thio-ATPA; 7, DNQX; 8, kainate; and 9, ATPO. * Figure 4: LBD conformational distributions in the context of an intact receptor. () Left, superposition of LBD conformations spanning the free energy landscapes onto the crystal structure of the intact GluA2 receptor (gray2; the ATD is not shown). The LBDs were superimposed only in lobe 1. Right, labeling of the four LBDs in an intact iGluR, as viewed from above. The LBDs assemble as a pair of dimers, where A–D is one dimer and B–C is the other. () R.m.s. displacement distributions of LBD conformations relative to the intact receptor. The r.m.s. displacement was measured in regions in lobe 2 (see Online Methods). The solid line indicates the average r.m.s. displacement (Supplementary Table 5). The dashed line indicates the r.m.s. displacement measured from the isolated LBD–ligand crystal structure. * Figure 5: Inter-LBD distance distributions. LBD conformations were superimposed onto the intact GluA2 structure (Fig. 4), and the pairwise distances were measured between regions in lobe 2 (see Online Methods). The apo LBD is gray, the LBD–antagonist complexes are blue and the LBD–full agonist complexes are red. The LBD–partial agonist complexes for kainate (), 4-AHCP () and thio-ATPA () are green. See Supplementary Table 5 for statistics and the distances measured using the isolated LBD–ligand crystal structures. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois, USA. * Albert Y Lau & * Benoît Roux * Present address: Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Albert Y Lau Contributions A.Y.L. and B.R. designed the research, analyzed the data and wrote the manuscript. A.Y.L. performed the computations. Competing financial interests The authors declare no competing financial interests. Author Details * Albert Y Lau Search for this author in: * NPG journals * PubMed * Google Scholar * Benoît Roux 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 Tables 1–5 and Supplementary Methods Additional data
• Competing allosteric mechanisms modulate substrate binding in a dimeric enzyme
  - Nat Struct Mol Biol 18(3):288-294 (2011)
  Nature Structural & Molecular Biology | Article Competing allosteric mechanisms modulate substrate binding in a dimeric enzyme * Lee A Freiburger1 * Oliver M Baettig2 * Tara Sprules3 * Albert M Berghuis2 * Karine Auclair1 * Anthony K Mittermaier1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:288–294Year published:(2011)DOI:doi:10.1038/nsmb.1978Received01 July 2010Accepted16 November 2010Published online30 January 2011 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 Allostery has been studied for many decades, yet it remains challenging to determine experimentally how it occurs at a molecular level. We have developed an approach combining isothermal titration calorimetry, circular dichroism and nuclear magnetic resonance spectroscopy to quantify allostery in terms of protein thermodynamics, structure and dynamics. This strategy was applied to study the interaction between aminoglycoside N-(6′)-acetyltransferase-Ii and one of its substrates, acetyl coenzyme A. It was found that homotropic allostery between the two active sites of the homodimeric enzyme is modulated by opposing mechanisms. One follows a classical Koshland-Némethy-Filmer (KNF) paradigm, whereas the other follows a recently proposed mechanism in which partial unfolding of the subunits is coupled to ligand binding. Competition between folding, binding and conformational changes represents a new way to govern energetic communication between binding sites. View full text Figures at a glance * Figure 1: Schematic representation of homotropic allosteric models for a dimeric protein. ○ and □ correspond to subunits in binding-incompetent and binding-competent states, respectively. () Monod-Wyman-Changeux (MWC) model: the symmetry of the dimer is preserved, so that only ○○ and □□ states are permitted. In the absence of ligand, both states are populated, and ligand binding forces the dimer into the □□ state. If the initial equilibrium favors the ○○ state, binding is positively cooperative, because the energetic cost of the ○○-to-□□ transition is paid by binding the first but not the second ligand. Note that in the standard MWC model, both ○○ and □□ bind ligand but with different affinities. For the sake of simplicity we have shown the limiting case where ○○ is binding-incompetent. () Koshland-Nemethy-Filmer (KNF) model: each subunit converts from the ○ to the □ state only upon binding ligand. Cooperativity is explained in terms of the strengths of subunit-subunit interactions. If the transition from the ○○ to ! the ○□ interface is energetically more favorable than that from ○□ to □□, binding is negatively cooperative, and the first ligand is bound more strongly than the second. If the transition from the ○○ to the ○□ interface is less favorable than from ○□ to □□, binding is positively cooperative, and the second ligand is bound more strongly than the first. () Hilser-Thompson (HT) model: in this case, ○ and □ correspond to the unfolded and folded states, respectively. Each unbound subunit can populate either the folded or unfolded state, and the folding equilibrium is influenced by the state of the adjacent monomer. If folding (□) of one subunit promotes folding (□) of the adjacent subunit, binding is positively cooperative. Conversely, if folding (□) of one subunit promotes unfolding (○) of the adjacent subunit, binding is negatively cooperative. * Figure 2: Temperature dependence of AAC(6′)-Ii binding thermodynamics and secondary structure. () Equilibrium association constants for the first and second molecules of AcCoA, plotted as a function of temperature. Solid lines correspond to the best fit obtained with equations (2) to (9). Dashed lines indicate the predicted affinities in the absence of thermal melting of the subunits. () Binding enthalpies of the first and second molecules of AcCoA. Inset shows the percentage of free subunits that are melted in the 0-bound and 1-bound forms. () Molar ellipticity (222 nm) of AAC(6′)-Ii as a function of temperature. Dashed and dash-dot lines correspond to the pre- and post-transition baselines, respectively. * Figure 3: Changes in AAC(6′)-Ii 800 MHz NMR spectra produced by AcCoA binding. (,) 1H-15N correlation spectra of AAC(6′)-Ii free () and saturated with AcCoA (). () X-ray crystal structure of the enzyme (PDB 2A4N25) bound to CoA (sticks), with blue spheres indicating the locations of residues with assigned cross-peaks in apo spectra. The backbone is color-coded according to the distance in the 1° sequence (n) from the nearest assigned residue according to 1 ≤ n ≤ 2 (light blue), 3 ≤ n ≤ 5 (white), 6 ≤ n ≤ 10 (pink), n > 10 (red). () Apparent minimal chemical shift differences ( ) between the free and bound states, mapped onto the X-ray crystal structure (PDB 2A4N25). Backbone amide nitrogen atoms are indicated with spheres for one subunit of the dimer and colored according to Δδapp < 0.5 p.p.m. (white), 0.5 ≤ Δδapp < 1 (light yellow), 1 ≤ Δδapp < 2 (yellow), 2 ≤ Δδapp < 4 (orange), 4 ≤ Δδapp (red). Unassigned residues, including prolines, are indicated with gray spheres. Structures were generated using PyMOL. * Figure 4: Analysis of NMR titration data. () Fraction of the enzyme in the 0-bound, 1-bound and 2-bound states determined by ITC. () Intensities of the apo (dashed line) and holo (solid line) peaks for Leu56 as a function of [AcCoA]. The intensities were analyzed to extract the relative contribution of the 1-bound enzyme to the signals ( ) as described in the text. The lines correspond to the optimized theoretical intensities. () Histograms of the relative contribution of the 1-bound enzyme to apo ( ) and holo ( ) peaks in titrations of AAC(6′)-Ii with AcCoA. * Figure 5: Schematic representation of the allosteric binding model. B corresponds to the bound state. F(F′) and U(U′) correspond to the free folded and partially unfolded states that are adjacent to a free (bound) subunit, respectively. Transitions involving the symmetry-related F′B and U′B states are not shown. * Figure 6: Dependence of the apparent cooperativity coefficient (αapp) on subunit instability (KU0 = [UF]/[FF]). (,) Shown are results of calculating this dependence using equation (1) and () the intrinsic cooperativity coefficient (αint = 1.3) and ratio of melting equilibrium constants (φ = 1.9) determined for AAC(6′)-Ii at 37 °C and () hypothetical proteins in which αint = φ = 4, 16, 64, 256, 1,024. The circle in corresponds to the KU0 value obtained for AAC(6′)-Ii at 37 °C (0.2). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 2A4N * 2A4N Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Chemistry, McGill University, Montréal, Québec, Canada. * Lee A Freiburger, * Karine Auclair & * Anthony K Mittermaier * Department of Biochemistry, McGill University, Montréal, Québec, Canada. * Oliver M Baettig & * Albert M Berghuis * Québec/Eastern Canada High Field NMR Facility, Montréal, Québec, Canada. * Tara Sprules Contributions L.A.F. and O.M.B. collected the data. L.A.F., O.M.B. and T.S. analyzed the data. A.K.M., K.A. and A.M.B. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Anthony K Mittermaier Author Details * Lee A Freiburger Search for this author in: * NPG journals * PubMed * Google Scholar * Oliver M Baettig Search for this author in: * NPG journals * PubMed * Google Scholar * Tara Sprules Search for this author in: * NPG journals * PubMed * Google Scholar * Albert M Berghuis Search for this author in: * NPG journals * PubMed * Google Scholar * Karine Auclair Search for this author in: * NPG journals * PubMed * Google Scholar * Anthony K Mittermaier Contact Anthony K Mittermaier 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 (800K) Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Methods Additional data
• Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating
  - Nat Struct Mol Biol 18(3):295-301 (2011)
  Nature Structural & Molecular Biology | Article Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating * Till Böcking1, 2, 5 * François Aguet2 * Stephen C Harrison3, 4 * Tomas Kirchhausen1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:295–301Year published:(2011)DOI:doi:10.1038/nsmb.1985Received13 July 2010Accepted23 November 2010Published online30 January 2011 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 Heat shock cognate protein-70 (Hsc70) supports remodeling of protein complexes, such as disassembly of clathrin coats on endocytic coated vesicles. To understand how a simple ATP-driven molecular clamp catalyzes a large-scale disassembly reaction, we have used single-particle fluorescence imaging to track the dynamics of Hsc70 and its clathrin substrate in real time. Hsc70 accumulates to a critical level, determined by kinetic modeling to be one Hsc70 for every two functional attachment sites; rapid, all-or-none uncoating then ensues. We propose that Hsc70 traps conformational distortions, seen previously by cryo-EM, in the vicinity of each occupied site and that accumulation of local strains destabilizes the clathrin lattice. Capture of conformational fluctuations may be a general mechanism for chaperone-driven disassembly of protein complexes. View full text Figures at a glance * Figure 1: A clathrin coat with views of a vertex before and after formation of an uncoating intermediate. () Schematic representation of clathrin triskelions in a D6-barrel lattice (PDB 1XI4; ref. 10). One clathrin triskelion is highlighted in blue. The green shaded leg segments show the invariant contact between proximal (p) and distal (d) legs of the triskelions indicated by green asterisks at their hubs. The green arrow shows the direction of conformational shift when auxilin and Hsc70 bind. The hook-like elements at the (N-terminal) tips of the legs represent the β-propeller terminal domains. () Detail of a vertex before binding of auxilin and Hsc70. The unstructured C termini of the clathrin heavy chain (blue balls), which contain the Q1638LMLT Hsc70-binding motif (narrow orange arrows), extend inward from the helical tripod at the triskelion hub14. The ankle (a) and terminal domain (t) shift in the direction of the wide green arrow when auxilin and Hsc70 bind. () Relative locations of bound auxilin (red spheres) and Hsc70 (orange lozenge) as determined by cryo-EM13, 14. T! he shift in the positions of the clathrin ankle and terminal domain have been exaggerated to illustrate the expansion of the funnel surrounding the Hsc70-binding motif. * Figure 2: Single-particle visualization of clathrin uncoating. () Schematic representation of the single-particle uncoating assay. The intensities of fluorescence from labeled clathrin and Hsc70 were monitored by TIRF microscopy for clathrin–AP-2 coats captured on the surface of a PEG-modified glass coverslip. () Representative time series of a single-particle uncoating assay. Left and right panels, first and last frames, respectively, of the fluorescence channel used to monitor the signal from coats tagged with clathrin LCa–AF488. Middle, kymograph generated from the vertical axis indicated by the arrows in the left panel, showing the unsynchronized disappearance of clathrin fluorescence. Hsc70–ATP (1.2 μM) arrived in the flow chamber at t = 0. () Uncoating profile from a single coat. The selected snapshots from the time series (top) show the fluorescence from clathrin and Hsc70 in the selected coat, at various time points during the uncoating reaction carried out with 0.9 μM Hsc70. The snapshots are background-corrected averag! es of three successive frames. The plot shows intensity traces of the clathrin (blue) and Hsc70 (orange) signals. The t = 0 time point is the moment at which a rapid increase in Hsc70 background signal is recorded; this event corresponds to the arrival of Hsc70 within the evanescent field at the coverslip. () Histogram of the number of trimers (triskelions) per coat at the beginning (top) and the end (bottom) of the single-particle uncoating assay carried out with 1.2 μM Hsc70. The number of trimers in intact coats follows a normal distribution with a mean of 34 triskelions per coat (top). In most cases, only one or two trimers remained at the site of a coat at the end of the reaction (bottom). Objects with overlapping point-spread functions were excluded from this analysis. A.u., arbitrary units. * Figure 3: Requirement of the Hsc70 binding motif for Hsc70-driven uncoating. () Representative uncoating traces for clathrin–AP-2 coats assembled with wild-type (WT) heavy chain clathrin (left panels) or mutant heavy chain clathrin truncated at its C terminus to remove the Hsc70 binding site (right panels). Hsc70–ATP (1.2 μM) arrived at t = 0 during monitoring of the fluorescence intensities of clathrin (blue) and Hsc70 (orange) with TIRF illumination. Vertical dotted lines indicate onset of coat disassembly. The Hsc70:triskelion ratios at the onset of coat disassembly for the WT traces are 1.0, 0.8, 1.2 and 0.6, from top to bottom. () Histogram of the time difference between maximal Hsc70 binding and onset of coat disassembly. () Histogram of the number of Hsc70 molecules bound per triskelion determined from the ratio of calibrated fluorescence intensities of Hsc70 to clathrin at the onset of coat disassembly (0.9 ± 0.3, N = 350 coats). See Online Methods for details. () Uncoating efficiency for coats assembled with either WT (N = 582 coats) o! r mutant clathrin (N = 660). * Figure 4: Hsc70 concentration dependence of the uncoating reaction. () Uncoating efficiency as a function of Hsc70 concentration. () Number of Hsc70 molecules bound per triskelion (mean ± s.d.) as a function of Hsc70 concentration determined from the ratio of the Hsc70 to the clathrin fluorescence intensity at the transition point between the accumulation and disassembly stages. * Figure 5: Recruitment of Hsc70 to clathrin–AP-2 coats during the accumulation phase follows first-order kinetics. The colored traces correspond to the averaged intensity of the Hsc70 fluorescence measurements recorded in separate, single-particle uncoating assays with various Hsc70 concentrations. We used a global fit of the accumulation phase up to the mean accumulation time for each concentration (black line) to determine the association and dissociation rates of Hsc70 binding. Mean accumulation times are derived from the accumulation time distributions in Figure 6. During this early recruitment phase, Hsc70 binds preferentially to the QLMLT sequence after activation by the neighboring auxilin J-domain. During the later stages, when most of the specific sites have been occupied, activated Hsc70 is presumably delivered to other sites, which do not drive disassembly. The average Hsc70 binding curves become noisy beyond their corresponding mean accumulation times because most coats have started to disassemble, and the average is calculated from an increasingly lower number of coats. The ! dashed lines show the binding curves from the calculated parameters, beyond the range of the data used for fitting. * Figure 6: Kinetic model for the uncoating reaction. () Scheme for the kinetic analysis of Hsc70-driven uncoating. The model assumes that a threshold number of sites in the coat, Nt, must be occupied by a productively bound Hsc70 molecule to initiate disassembly. Hsc70 binds independently to the N binding positions in the coat (N = 36 for the D6 barrel), with a microscopic association rate constant k1+ (determined from the Hsc70 association curves; see Supplementary Methods and Fig. 5). The rate of any step is given by the product of k1+ and the number of unoccupied sites. Bound Hsc70 dissociates from the coat with an off-rate given by the microscopic dissociation rate constant k1− and the number of bound Hsc70 molecules. Binding of Nt Hsc70 molecules triggers disassembly in a single, rate-limiting step of rate constant k2+ (determined independently; see Fig. 7). The rate of clathrin dissociation, which depends linearly on Hsc70 concentration, is modeled with a single rate constant, k3+. () Distributions of the Hsc70 accumul! ation time (pink) and the full uncoating time (cyan), comprising accumulation and disassembly phases, at various Hsc70 concentrations, overlaid with fits of the kinetic model. The insets show disassembly time distributions (green) at various Hsc70 concentrations and the corresponding single exponential phase derived from the model fit. * Figure 7: Transition of Hsc70-loaded coats to the disassembly phase. () Experimental design used for the pH shift experiment. After immobilization of the clathrin–AP-2 coats on the modified coverslip, the microfluidic chamber was perfused sequentially with auxilin and then with a mixture of auxilin and Hsc70–AF568–ATP at pH 6. At this pH, the coats do not dissociate23. To trigger uncoating, the pH was shifted to 6.8 in the presence of unlabeled Hsc70–ATP. Minimal uncoating was observed if the pH shift was done in the absence of Hsc70–ATP. () Histogram of the dwell times between pH shift and initiation of disassembly. The distribution of dwell times is a single exponential, indicating that the transition of an Hsc70-loaded coat to the disassembly phase has a single rate-limiting step, with rate constant k2+ = 0.16 s−1. * Figure 8: Uncoating reaction with coats containing mixtures of wild-type and mutant clathrin. () Backbone model of the heavy and light chains of a clathrin triskelion (PDB 3IYV)10. The close-up of a triskelion hub shows the location of the binding site for Hsc70 (QLMLT motif, black dots) in the C-terminal unstructured region of the clathrin heavy chain (HC; blue lines). (,) Outcome of the uncoating reaction for coats containing mostly wild-type (WT) () or mostly mutant clathrin lacking the Hsc70-binding motif (). Coats containing primarily WT clathrin underwent normal disassembly with release of WT and mutant clathrin (left); coats containing primarily mutant clathrin remained intact, and no WT clathrin was released. Top, schematic representation of the coats and the outcome of the uncoating reaction. Middle, representative kymographs of single-particle uncoating traces from two coats, one containing excess WT clathrin (80%, left) and the other containing excess mutant clathrin (80%, right); WT and mutant clathrin were labeled with LCa–AF488 and LCa–DL649, respec! tively. Bottom, plots of fluorescence intensity traces: blue, WT clathrin; purple, mutant clathrin; orange, Hsc70. () Uncoating efficiency as a function of the fraction of WT clathrin in the coat. Coats with different ratios of WT and mutant clathrin were prepared and mixed; their disassembly properties were then analyzed together in the same field (N = 311 coats). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 1XI4 * 3IYV * 1XI4 * 3IYV Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Immune Disease Institute, Harvard Medical School, Boston, Massachusetts, USA. * Till Böcking & * Tomas Kirchhausen * Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA. * Till Böcking, * François Aguet & * Tomas Kirchhausen * Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA. * Stephen C Harrison * Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA. * Stephen C Harrison * Centre for Vascular Research, University of New South Wales, Australia. * Till Böcking Contributions T.B. and T.K. developed the single-molecule assay and T.B. performed experiments; T.B. and F.A. analyzed data; F.A. developed the kinetic model. T.B., S.C.H. and T.K. designed experiments. All authors discussed the results and contributed to the final manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Tomas Kirchhausen Author Details * Till Böcking Search for this author in: * NPG journals * PubMed * Google Scholar * François Aguet Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen C Harrison Search for this author in: * NPG journals * PubMed * Google Scholar * Tomas Kirchhausen Contact Tomas Kirchhausen 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 (8M) Supplementary Figures 1–12 and Supplementary Methods Additional data
• LIN-28 co-transcriptionally binds primary let-7 to regulate miRNA maturation in Caenorhabditis elegans
  - Nat Struct Mol Biol 18(3):302-308 (2011)
  Nature Structural & Molecular Biology | Article LIN-28 co-transcriptionally binds primary let-7 to regulate miRNA maturation in Caenorhabditis elegans * Priscilla M Van Wynsberghe1 * Zoya S Kai1 * Katlin B Massirer2, 3, 4 * Victoria H Burton1 * Gene W Yeo2, 3, 4 * Amy E Pasquinelli1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:302–308Year published:(2011)DOI:doi:10.1038/nsmb.1986Received15 May 2010Accepted23 November 2010Published online06 February 2011 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 highly conserved let-7 microRNA (miRNA) regulates developmental pathways across animal phyla. Mis-expression of let-7 causes lethality in C. elegans and has been associated with several human diseases. We show that timing of let-7 expression in developing worms is under complex transcriptional and post-transcriptional control. Expression of let-7 primary transcripts oscillates during each larval stage, but precursor and mature let-7 miRNAs do not accumulate until later in development after LIN-28 protein has diminished. We demonstrate that LIN-28 binds endogenous primary let-7 transcripts co-transcriptionally. We further show that LIN-28 binds endogenous primary let-7 transcripts in the nuclear compartment of human ES cells, suggesting that this LIN-28 activity is conserved across species. We conclude that co-transcriptional interaction of LIN-28 with let-7 primary transcripts blocks Drosha processing and, thus, precocious expression of mature let-7 during early developm! ent. View full text Figures at a glance * Figure 1: Expression of let-7 is transcriptionally and post-transcriptionally regulated. () Depiction of the 2,460-nt-long let-7 rescue construct with the positions of the mature let-7 sequence (blue), 3′ splice site (SS; yellow) and two start sites (A and B) and the approximate sizes of the spliced and unspliced transcripts indicated4, 7. () Northern blot analysis of total RNA isolated from embryos (E) or synchronized plet-7B∷GFP transgenic worms. The similar-sized B and SL1 transcripts often do not clearly resolve. () Agarose or PAGE northern blot analysis of total RNA isolated from embryos (E) or synchronized WT N2 worms at larval (L) and adult (AD) stages. Representative blots from four independent experiments are shown. () Average levels of pri-, pre- and mature let-7 after normalization to 18S or 5.8S rRNA from four independent experiments. () Analysis as in of total RNA isolated from synchronized plet-7B∷GFP transgenic worms. The entire blot is shown in Supplementary Figure 1c. * Figure 2: Developmentally regulated processing of let-7 pri-miRNA transcripts. () Depiction of expected Drosha cleavage products: 5′ flanking, let-7 hairpin precursor and 3′ flanking. The number of sequenced RACE clones that mapped to the precise 3′ and 5′ Drosha cleavage products at each time point from two independent experiments is shown. The sequences of all Drosha cleavage products are shown in Supplementary Figure 3. () RT-PCR analysis of two independent 5′ RACE samples from N2 (left) or N2 and lin-28(n719) worms (right). () Agarose and PAGE northern blot analysis of total RNA isolated from synchronized eri-1(mg366) RNAi-hypersensitive worms at the indicated time points after vector control (−) or pup-2 (+) RNAi treatment. Representative blots from three independent experiments are shown. * Figure 3: Regulation of let-7 processing by LIN-28. () Agarose and PAGE northern blotting analysis of total RNA isolated from WT N2 or lin-28(n719) embryos (E) and synchronized worms at the indicated time points. Representative blots from three independent experiments are shown. The arrowheads mark the location of the SL1 pri-let-7 transcript. () Analysis as in of total RNA from late L1 and early L2. Representative blots from three independent experiments are shown. (,) Levels of each pri-let-7 isoform at the 10-h time point in lin-28(n719) relative to WT N2 worms after normalization of 18S rRNA, calculated from six independent northern blot experiments () or three independent qRT-PCR experiments () and analyzed by Student's t-test (*P < 0.05, **P < 0.005, ***P < 0.0005). Error bars, s.e.m. * Figure 4: LIN-28 binds endogenous let-7 primary transcripts in C. elegans and human ES cells. (,) Western blot analysis of total protein isolated from PQ272 (LIN-28:GFP) worms. () Ratios of LIN-28:GFP levels to the 10-h time point after tubulin normalization, calculated from three independent experiments and analyzed by Student's t-test (***P < 0.0005). Error bars, s.e.m. () RNA immunoprecipitation (RIP) analysis of synchronized PQ272 worms collected at 10 h. Input, and LIN-28:GFP and IgG immunoprecipitates, were analyzed by western blotting or RT-PCR. α, antibody. () RIP analysis of undifferentiated HUES6 cells. Input, LIN-28 and IgG immunoprecipitates were analyzed by western blotting or RT-PCR. () qRT-PCR analysis of the worm and human cell samples from , to determine the levels of input or LIN-28 immunoprecipitated pri- or pre-let-7 RNAs using primers specific for pri-let-7 (priF and priR) or pre-let-7 and pri-let-7 transcripts containing the precursor hairpin (preF and preR). The ratio of precursor containing let-7 transcripts to pri-let-7 transcripts for immun! oprecipitated samples after normalization to input samples for at least three independent experiments is shown, and was analyzed by Student's t-test (*P < 0.05, **P < 0.005, ***P < 0.0005). Error bars, s.e.m. (,) RIP and western blotting analysis () or qRT-PCR analysis as in () of undifferentiated HUES6 cells fractionationated into nuclear and cytoplasmic extracts. Results from three independent experiments are shown. RNAP II, RNA polymerase II. * Figure 5: LIN-28 binds endogenous let-7 genomic DNA. Chromatin immunoprecipitation (ChIP) analysis of synchronized PQ272 (LIN-28:GFP) or pD4792 (GFP) worms collected at 10 h. ChIP was carried out with polyclonal antibodies against RNA polymerase II (αPoI II) or GFP (αGFP) or with IgG. The ratio of the indicated genomic DNA in the immunoprecipitated sample to the input sample for at least three independent experiments is shown and was analyzed by Student's t-test (*P < 0.05). Error bars, s.e.m. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Biology, University of California, San Diego, La Jolla, California, USA. * Priscilla M Van Wynsberghe, * Zoya S Kai, * Victoria H Burton & * Amy E Pasquinelli * Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USA. * Katlin B Massirer & * Gene W Yeo * Stem Cell Program, University of California, San Diego, La Jolla, California, USA. * Katlin B Massirer & * Gene W Yeo * Institute for Genomic Medicine, University of California, San Diego, La Jolla, California, USA. * Katlin B Massirer & * Gene W Yeo Contributions A.E.P. and P.M.V. designed the project and wrote the paper; P.M.V. (all figures), Z.S.K. (Fig. 1 and Supplementary Fig. 1), K.B.M. (Fig. 4) and V.H.B. (Fig. 4) performed the experiments; A.E.P. and G.W.Y. supervised the studies. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Amy E Pasquinelli Author Details * Priscilla M Van Wynsberghe Search for this author in: * NPG journals * PubMed * Google Scholar * Zoya S Kai Search for this author in: * NPG journals * PubMed * Google Scholar * Katlin B Massirer Search for this author in: * NPG journals * PubMed * Google Scholar * Victoria H Burton Search for this author in: * NPG journals * PubMed * Google Scholar * Gene W Yeo Search for this author in: * NPG journals * PubMed * Google Scholar * Amy E Pasquinelli Contact Amy E Pasquinelli 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 1–7, Supplementary Tables 1–4 and Supplementary Methods Additional data
• Structural insights into energy regulation of light-harvesting complex CP29 from spinach
  - Nat Struct Mol Biol 18(3):309-315 (2011)
  Nature Structural & Molecular Biology | Article Structural insights into energy regulation of light-harvesting complex CP29 from spinach * Xiaowei Pan1 * Mei Li1 * Tao Wan1, 2 * Longfei Wang1, 2 * Chenjun Jia1, 2 * Zhiqiang Hou1, 2 * Xuelin Zhao1 * Jiping Zhang1 * Wenrui Chang1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:309–315Year published:(2011)DOI:doi:10.1038/nsmb.2008Received28 July 2010Accepted23 November 2010Published online06 February 2011 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 CP29, one of the minor light-harvesting complexes of higher-plant photosystem II, absorbs and transfers solar energy for photosynthesis and also has important roles in photoprotection. We have solved the crystal structure of spinach CP29 at 2.80-Å resolution. Each CP29 monomer contains 13 chlorophyll and 3 carotenoid molecules, which differs considerably from the major light-harvesting complex LHCII and the previously proposed CP29 model. The 13 chlorophyll-binding sites are assigned as eight chlorophyll a sites, four chlorophyll b and one putative mixed site occupied by both chlorophylls a and b. Based on the present X-ray structure, an integrated pigment network in CP29 is constructed. Two special clusters of pigment molecules, namely a615–a611–a612–Lut and Vio(Zea)–a603–a609, have been identified and might function as potential energy-quenching centers and as the exit or entrance in energy-transfer pathways. View full text Figures at a glance * Figure 1: Stereo view of the overall structure of CP29. View in parallel with the membrane plane. Helices A–E are labeled in the same way as they are in spinach LHCII (PDB code 1RWT)4. For clarity, the Chl phytyl chains are not shown. Green, Chl a; blue, Chl b; yellow, Lut; orange, Vio; magenta, Neo; light pink, G3P. * Figure 2: Structural comparison between CP29 and LHCII (PDB 1RWT)4. () Superposition of the two structures based on Cα of their helices C. View along the membrane plane. Both the apoprotein and pigments of LHCII are shown in gray. The apoprotein of CP29 is shown as a yellow ribbon, and its pigments are shown as ball-and-stick models with the same color designation as in Figure 1. Conserved pigments in the two structures are not shown in this figure. (,) Comparison of Chl 609 binding site in CP29 () and LHCII (). The hydrogen bonds are indicated by dark dotted lines. The amide side chain of Gln131 in LHCII interacts with three Chls b. One hydrogen bond is formed through the interactions of its C=O bond with the coordinated water of Chl b606, and two are formed by NH2 with the C7-formyls of Chls b607 and b609. In CP29, however, the corresponding residue is Glu151, with no additional hydrogen in its side chain for hydrogen bond formation with Chl a609. (,) Comparison of Chl 614 binding site in CP29 () and LHCII (). In CP29, NH of Trp226 side c! hain forms a hydrogen bond with Chl b614 C7-formyl, whereas in LHCII, the corresponding residue is Leu209 with no hydrogen bond interaction with Chl a614. * Figure 3: Room temperature (20 °C) absorption spectra of CP29 showing its characteristic features. Black line, absorption spectrum of CP29 protein used for crystallization; black dotted line, absorption spectrum of CP29 crystal. * Figure 4: Pigment arrangement in CP29. () Arrangement of Chls. View along the membrane plane. Chls are represented by three atoms: the central magnesium atom and two nitrogen atoms (NB, ND). Yellow, Chl a magnesium; white, Chl b magnesium; green, Chl a nitrogen; blue, Chl b nitrogen. The adjacent Chls in each layer, the closest Chls from the two layers and the closest Chls from the two clusters (Chls a604–b606–b607 and Chls a613–b614) in the lumenal layer are connected with dark dashed lines. Their center-to-center distances (Å) are labeled with red numbers. The coordinates of molecular centers of Chls are calculated by arithmetically averaging the coordinates of the four pyrrole nitrogen atoms. (–) Lut, Vio, Neo and their neighboring Chls. () Superposition of Lut and Vio showing similar arrangements with their neighboring Chls a. Green, Chls a around Lut; gray, Chls a around Vio. Nitrogen atoms NB and ND in each Chl a are shown in blue and magenta, respectively. * Figure 5: Strongly interacting pigment cluster a615–a611–a612–Lut. (,) Chl pair of a611–a615. The hydrogen bonds are connected with dark dotted lines. The ionic bond between Lys199 and G3P is indicated by a blue dotted line (). The Qy transition dipole moments of Chls a611 and a615 are marked with green and cyan arrows, respectively (). () Stereo view of pigment cluster a615–a611–a612–Lut. The numerical note near the dark dashed line connecting two pigments indicates the center-to-center distance (Å) between them. * Figure 6: Pigment cluster Vio(Zea)–a603–a609. () 2Fo – Fc (1.5σ level, cyan) and Fo – Fc (3.5σ level, magenta) electron density of Vio. Strong 2Fo – Fc and Fo – Fc densities show up at the position of epoxy groups in the two end rings, if they are deleted. () Vio binding site and the pigment cluster Vio(Zea)–a603–a609. A hydrogen bond network is formed through the interaction of the hydroxyl in Vio lumenal end ring with the carboxyl of Trp121, as well as the interaction of a water molecule with the coordinated water of Chl a604. Water molecules are shown as cyan spheres. The numerical note near the dark dashed line connecting two pigments indicates the center-to-center distance (Å) between them. () The local environment of Vio(Zea)–a603–a609. The acidic residues in the lumenal side are shown as a magenta surface. The polar residues around the pigment cluster are shown as a yellow surface. Vio is represented by orange spheres. Chls a603 and a609 are represented by green and cyan ball-and-stick models. ! Other Chls (a602, a604 and b606, b607) forming a wide range of van der Waals interactions with Vio are shown as lines. The color designation is the same as in Figure 1. * Figure 7: Two important pigment clusters in CP29. () View along the membrane plane. () View from the stromal side. Pigment clusters a615–a611–a612–Lut and Vio(Zea)–a603–a609 are shown as sticks. Other pigments are shown as lines with the same color designation as in Figure 1. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3PL9 * 3PL9 Referenced accessions Protein Data Bank * 1RWT * 2BHW * 1RWT * 2BHW Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. * Xiaowei Pan, * Mei Li, * Tao Wan, * Longfei Wang, * Chenjun Jia, * Zhiqiang Hou, * Xuelin Zhao, * Jiping Zhang & * Wenrui Chang * Graduate University of the Chinese Academy of Sciences, Beijing, China. * Tao Wan, * Longfei Wang, * Chenjun Jia & * Zhiqiang Hou Contributions X.W.P. did the purification, crystallization, data collection and processing, structure determination and structural analysis. M.L. assisted in data collection, structure analysis and manuscript preparation. T.W. did the protein sequence determination. L.F.W. assisted in data collection and structure determination. C.J.J., Z.Q.H., X.L.Z. and J.P.Z. assisted in sample isolation and purification. W.R.C. supervised the project and analyzed the structure. X.W.P. and W.R.C. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Wenrui Chang Author Details * Xiaowei Pan Search for this author in: * NPG journals * PubMed * Google Scholar * Mei Li Search for this author in: * NPG journals * PubMed * Google Scholar * Tao Wan Search for this author in: * NPG journals * PubMed * Google Scholar * Longfei Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Chenjun Jia Search for this author in: * NPG journals * PubMed * Google Scholar * Zhiqiang Hou Search for this author in: * NPG journals * PubMed * Google Scholar * Xuelin Zhao Search for this author in: * NPG journals * PubMed * Google Scholar * Jiping Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Wenrui Chang Contact Wenrui Chang 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 (3M) Supplementary Figures 1–5 and Supplementary Tables 1–3 Additional data
• Structural basis for allosteric regulation of human ribonucleotide reductase by nucleotide-induced oligomerization
  - Nat Struct Mol Biol 18(3):316-322 (2011)
  Nature Structural & Molecular Biology | Article Structural basis for allosteric regulation of human ribonucleotide reductase by nucleotide-induced oligomerization * James Wesley Fairman1, 9 * Sanath Ranjan Wijerathna2, 9 * Md Faiz Ahmad2 * Hai Xu2, 8 * Ryo Nakano4 * Shalini Jha2 * Jay Prendergast2 * R Martin Welin5 * Susanne Flodin5 * Annette Roos5 * Pär Nordlund5 * Zongli Li6 * Thomas Walz6 * Chris Godfrey Dealwis2, 3, 7 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:316–322Year published:(2011)DOI:doi:10.1038/nsmb.2007Received23 June 2010Accepted30 November 2010Published online20 February 2011 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 Ribonucleotide reductase (RR) is an αnβn (RR1–RR2) complex that maintains balanced dNTP pools by reducing NDPs to dNDPs. RR1 is the catalytic subunit, and RR2 houses the free radical required for catalysis. RR is allosterically regulated by its activator ATP and its inhibitor dATP, which regulate RR activity by inducing oligomerization of RR1. Here, we report the first X-ray structures of human RR1 bound to TTP alone, dATP alone, TTP–GDP, TTP–ATP, and TTP–dATP. These structures provide insights into regulation of RR by ATP or dATP. At physiological dATP concentrations, RR1 forms inactive hexamers. We determined the first X-ray structure of the RR1–dATP hexamer and used single-particle electron microscopy to visualize the α6–ββ′–dATP holocomplex. Site-directed mutagenesis and functional assays confirm that hexamerization is a prerequisite for inhibition by dATP. Our data indicate a mechanism for regulating RR activity by dATP-induced oligomerization. View full text Figures at a glance * Figure 1: Structure of hRRM1. () Ribbon diagram of hRRM1 dimer. Chains A and B are yellow and cyan, respectively. The four-helix ATP-binding cones of both subunits are red. TTP (green), GDP (orange), and ATP or dATP (purple) bound at the S, C and A sites, respectively, are transparent surfaces. () The four-helix cone with ATP bound. 2Fo – Fc electron density for ATP (carbon, oxygen and nitrogen atoms are yellow, red and blue, respectively) contoured at 1 σ is in green wire mesh. () The four-helix cone with dATP bound. 2Fo – Fc electron density for dATP (carbon, oxygen and nitrogen atoms are black, red and blue, respectively) contoured at 1 σ is in blue wire mesh. * Figure 2: ATP and dATP binding at the A site of hRRM1. All panels are stereoviews. () ATP binding. The protein is represented as ribbons. Carbon, oxygen and nitrogen atoms are cyan, red and blue, respectively. Interacting residues are sticks, and ATP is represented as sticks with yellow carbon and phosphorous atoms. Hydrogen bonds and electrostatic interactions are black dashed lines. Magnesium atoms are green spheres. () dATP binding. The protein is represented as ribbons with carbons in magenta, and interacting residues are sticks. dATP is represented as sticks with its carbon and phosphorous atoms in green. Hydrogen bonds and electrostatic interactions are dashed black lines. () The ATP-binding cones of hRRM1–TTP–ATP (blue) and hRRM1–TTP–dATP (magenta) were aligned to that of hRRM1–TTP (orange). The proteins are ribbons, the ATP nucleotide is yellow and the entire dATP nucleotide is green. () Comparison of ATP and dATP binding by superposing and . The same coloring scheme as in and is used. * Figure 3: SEC analysis of hRRM1 oligomers and enzyme activities of wild-type and mutant hRRM1. () The protein concentration of hRRM1 was 1.25 μM. hRRM1 forms monomers without dATP (blue trace) and a mixed population of monomers, dimers and hexamers at 5 μM dATP (red trace). At 20 μM dATP, the hexamers are the dominant species, with a small amount of dimer (green trace). () Standard curve for determination of molecular masses (Mr) of RR. Kav = (Ve – Vo)/(Vt – Vo), where Ve = elution volume, Vo = void volume and Vt = total volume. () SEC analysis of RR holocomplex with t-hRRM1 and hRRM2. The t-hRRM1 in the presence of 20 μM dATP formed a dimer that eluted at a molecular mass of 186 kDa. When the two species were mixed, they eluted at a molecular mass of 278 kDa, corresponding to an α2β2 holocomplex. () Wild-type (green trace), D16R (purple trace) and H2E (orange trace) hRRM1 proteins at 10 μM were tested for their ability to form hexamers in the presence of 20 μM dATP. () The specific activity of wild-type and mutant hRRM1 was determined using [3H]CDP and [1! 4C]ADP reduction assays. () Specific activity of D16R in the presence of 20 μM dATP. [3H]CDP reduction was carried out in the presence of 3 mM ATP and with and without 20 μM dATP. When [14C]ADP was used as the substrate, D16R activity was measured without ATP and with and without 20 μM dATP. Error bars, s.d. * Figure 4: Hexameric packing of RR1 based on the low-resolution X-ray crystal structure of the ScRR1 hexamer. (,) Ribbon diagrams of the two possible hexamer packing arrangements. ScRR1 monomers are dark and light green or blue and cyan. All of the four-helix ATP-binding cones are red. () Model of the RR holocomplex based on the ScRR1 hexamer in and the positions of the ScRR2 subunits modeled based on the StRR1–StRR2 holocomplex. ScRR1 monomers are dark and light green with the ATP-binding cones in red. ScRR2 subunits are light and dark purple. () Model of the RR holohexamer complex based on the ScRR1 hexamer in and the positions of the ScRR2 subunits modeled based on the StRR1–StRR2 holocomplex. ScRR1 monomers are blue and cyan with the ATP-binding cones in red. ScRR2 subunits are light and dark purple. * Figure 5: Electron microscopy of the α6–ββ′–dATP holocomplex. () Raw image of the holocomplex in negative stain. Scale bar, 50 nm. Right panels, representative class averages. Side length of individual panels, 35 nm. () Different views of the 28-Å density map calculated using the random conical tilt approach with 50°/0° image pairs of cryonegatively stained holocomplex. Scale bar, 5 nm. () Model of α6–ββ′–dATP holocomplex. Gray contour, crystal structure of RR1–dATP hexamer resolution-filtered to 28 Å. Golden contour, difference density obtained by subtracting density of RR1–dATP hexamer from EM density map of holocomplex. Green ribbon diagram, RR1–dATP hexamer; red ribbon diagram, yeast ββ′ heterodimer (PDB 1JKO)42 docked into the difference peak. () Model for dATP-dependent oligomerization of eukaryotic RRs. Binding of effectors to the S site causes dimerization, and binding of dATP to the A site causes the formation of hexamers via a hypothesized short-lived tetramer intermediate or the immediate association o! f three dimers to form a hexamer (question mark). Effectors bound at the S site are purple spheres, and dATPs bound at the A site are blue spheres. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3HNC * 2WGH * 3HND * 3HNE * 3HNF * 3PAW * 3HNC * 2WGH * 3HND * 3HNE * 3HNF * 3PAW Referenced accessions Protein Data Bank * 1JKO * 1JKO Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * James Wesley Fairman & * Sanath Ranjan Wijerathna Affiliations * Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee, USA. * James Wesley Fairman * Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA. * Sanath Ranjan Wijerathna, * Md Faiz Ahmad, * Hai Xu, * Shalini Jha, * Jay Prendergast & * Chris Godfrey Dealwis * Center for Proteomics and Bioinformatics at Case Western Reserve University, Cleveland, Ohio, USA. * Chris Godfrey Dealwis * Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA. * Ryo Nakano * Structural Genomics Consortium, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. * R Martin Welin, * Susanne Flodin, * Annette Roos & * Pär Nordlund * Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA. * Zongli Li & * Thomas Walz * Department of Chemistry, Case Western Reserve University, Cleveland, Ohio, USA. * Chris Godfrey Dealwis * Present address: R&D Lab, Trevigen, Gaithersburg, Maryland, USA. * Hai Xu Contributions J.W.F. purified protein, collected X-ray data and determined hRR1 and ScRR1 structures. S.R.W. purified protein, crystallized the hexamer, collected X-ray data, and did all the biochemical experiments. M.F.A. conducted the CD and fluorescence experiments. H.X. collected data and determined hexamer structure. R.N. and S.J. helped in protein purification. J.P. helped with SEC. R.M.W., S.F. and A.R. conducted crystallography and SEC studies of truncated hRR1. P.N. directed research on the truncated hRR1. Z.L. collected EM data and did image reconstruction. T.W. directed EM experiments. J.W.F., S.R.W., M.F.A., P.N. and T.W. helped with manuscript preparation. C.G.D. directed research, data analysis and manuscript preparation. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Chris Godfrey Dealwis Author Details * James Wesley Fairman Search for this author in: * NPG journals * PubMed * Google Scholar * Sanath Ranjan Wijerathna Search for this author in: * NPG journals * PubMed * Google Scholar * Md Faiz Ahmad Search for this author in: * NPG journals * PubMed * Google Scholar * Hai Xu Search for this author in: * NPG journals * PubMed * Google Scholar * Ryo Nakano Search for this author in: * NPG journals * PubMed * Google Scholar * Shalini Jha Search for this author in: * NPG journals * PubMed * Google Scholar * Jay Prendergast Search for this author in: * NPG journals * PubMed * Google Scholar * R Martin Welin Search for this author in: * NPG journals * PubMed * Google Scholar * Susanne Flodin Search for this author in: * NPG journals * PubMed * Google Scholar * Annette Roos Search for this author in: * NPG journals * PubMed * Google Scholar * Pär Nordlund Search for this author in: * NPG journals * PubMed * Google Scholar * Zongli Li Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Walz Search for this author in: * NPG journals * PubMed * Google Scholar * Chris Godfrey Dealwis Contact Chris Godfrey Dealwis 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 Discussion (including three figures and two tables), Supplementary Methods (including six figures and two tables), Supplementary Tables 1 and 2, and Supplementary Figures 1–5 Additional data
• Competition for XPO5 binding between Dicer mRNA, pre-miRNA and viral RNA regulates human Dicer levels
  - Nat Struct Mol Biol 18(3):323-327 (2011)
  Nature Structural & Molecular Biology | Article Competition for XPO5 binding between Dicer mRNA, pre-miRNA and viral RNA regulates human Dicer levels * Yamina Bennasser1 * Christine Chable-Bessia1 * Robinson Triboulet1 * Derrick Gibbings2 * Carole Gwizdek3 * Catherine Dargemont3 * Eric J Kremer4 * Olivier Voinnet2 * Monsef Benkirane1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:323–327Year published:(2011)DOI:doi:10.1038/nsmb.1987Received11 February 2010Accepted23 November 2010Published online06 February 2011 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 MicroRNAs (miRNAs) are a class of small, noncoding RNAs that function by regulating gene expression post-transcriptionally. Alterations in miRNA expression can strongly influence cellular physiology. Here we demonstrated cross-regulation between two components of the RNA interference (RNAi) machinery in human cells. Inhibition of exportin-5, the karyopherin responsible for pre-miRNA export, downregulated expression of Dicer, the RNase III required for pre-miRNA maturation. This effect was post-transcriptional and resulted from an increased nuclear localization of Dicer mRNA. In vitro assays and cellular RNA immunoprecipitation experiments showed that exportin-5 interacted directly with Dicer mRNA. Titration of exportin-5 by overexpression of either pre-miRNA or the adenoviral VA1 RNA resulted in loss of Dicer mRNA–exportin-5 interaction and reduction of Dicer level. This saturation also occurred during adenoviral infection and enhanced viral replication. Our study reveals ! an important cross-regulatory mechanism between pre-miRNA or viral small RNAs and Dicer through exportin-5. View full text Figures at a glance * Figure 1: Regulation of Dicer protein level by XPO5. () Expression of XPO5, Dicer, Drosha and DDX6 in HeLa cells transfected with control scrambled (Scr) or specific siRNA, as determined by western blotting. () Expression of Dicer, Myc-XPO5 and tubulin in HeLa cells transfected with either Myc-XPO5 or empty expression vectors, as determined by western blotting. () Dicer mRNA levels of samples described in , as analyzed by qRT-PCR after total RNA extraction. Error bars, s.d. * Figure 2: XPO5 knockdown results in accumulation of Dicer mRNA in the nucleus. Quantification of Dicer mRNA, U6 small nuclear RNA and GAPDH mRNA by qRT-PCR in nuclear and cytoplasmic fractionated RNA from HeLa cells transfected with XPO5-specific siRNA or control scrambled siRNA. Cytoplasmic RNAs were normalized to GAPDH mRNA and nuclear RNAs to U6 snRNA. Results are expressed in arbitrary units and are representative of three independent experiments. Error bars, s.d. * Figure 3: Dicer mRNA specifically interacts with XPO5 in vivo and in vitro. () HeLa cell extracts were subjected to immunoprecipitation using IgG (control) or antibodies to CRM1, XPO5 or TAP–p15 in the presence of RanQ69L-GTP. Fractions of the unbound (FT) material and immunoprecipitates (IPs) were analyzed by western blotting using specific antibodies (Supplementary Fig. 2), and the rest of the IPs were used for RNA extraction. Purified RNA were reverse transcribed and subjected to qRT-PCR using specific primers for U3 snoRNA, pre-miR-16, GAPDH mRNA and Dicer mRNA. () Electrophoretic mobility shift assay using a radiolabeled Dicer mRNA probe (see Online Methods). Complex was formed in the presence of increasing amounts of recombinant XPO5 and RanQ69L-GTP (lanes 1–7). Complexes were subjected to treatment with RNase and analyzed on nondenaturing 5% acrylamide/TBE gels. Increasing amounts of unlabeled pre-miR-30 (10, 50, 250 ng) were used as specific competitor. Both binding assays were carried out in the presence of RanQ69L-GTP to increase speci! ficity (Supplementary Fig. 3). () Radiolabeled pre-miR-30 was incubated with 200 ng of XPO5 in the presence of RanQ69L-GTP as described in Online Methods. Complexes were formed in the absence (−) or in the presence of increasing amounts (0.5, 1 and 2 μg) of the competitor RNAs as indicated. Note that this EMSA is done in the absence of RNase treatment. Complexes were analyzed on nondenaturing 5% acrylamide/TBE gels. * Figure 4: Overexpression of pre-miRNA or adenoviral VA1 RNA affects Dicer protein levels in cells. () HeLa cells were transfected with XPO5- or Dicer-specific siRNA, empty vector, or increasing amounts of a vector expressing pre-miR-30. Dicer and tubulin expression were analyzed by western blot (left), CRM1, XPO5 or TAP–p15 were immunoprecipitated from cell extracts and associated RNAs were extracted. Recovered RNAs were subjected to qRT-PCR to quantify U3 snoRNA, U6 snRNA, pre-miR-16 and GAPDH mRNA, as controls (Supplementary Fig. 4), as well as Dicer mRNA. () As in except that cells were transfected with either empty vector (pVV2) or increasing amounts of plasmid expressing VA1 RNA (pVA1). * Figure 5: XPO5 inhibition enhances adenovirus replication. (,) HeLa cells were infected with wild-type (Ad5) or Ad720 mutant (dlsub720) adenovirus. Infected cells were harvested at different times after infection. A fraction of each cell extract was analyzed for viral capsid, Dicer, XPO5 and tubulin by western blotting () and the remainder was subjected to RNA purification. Dicer and GAPDH mRNAs (, left), U6 snRNA and VA1 RNA (, right) were quantified by qRT-PCR. (,) Top panels show knockdown efficiency of XPO5 () and Dicer () in HeLa cells. siRNA-transfected HeLa cells were infected with Ad5 or Ad720 (0.1 particle per cell). Cells were harvested every 12 h up to 48 h after infection () or just at 48 h after infection (). Bottom panels show levels of adenoviral DNA as quantified by qPCR using primers amplifying the DBP viral gene present in both viruses. Results are expressed after normalization with respect to GAPDH. Error bars, s.d. Author information * Abstract * Author information * Supplementary information Affiliations * Centre National de la Recherche Scientifique (CNRS), Institut de Génétique Humaine UPR1142, Laboratoire de Virologie Moléculaire, Montpellier, France. * Yamina Bennasser, * Christine Chable-Bessia, * Robinson Triboulet & * Monsef Benkirane * Institut de Biologie Moléculaire des Plantes (IBMP), CNRS, Université de Strasbourg, Strasbourg, France. * Derrick Gibbings & * Olivier Voinnet * Institut Jacques Monod, Université Paris Diderot, CNRS, Paris, France. * Carole Gwizdek & * Catherine Dargemont * Institut de Génétique Moléculaire de Montpellier, Universités de Montpellier I & II, Montpellier, France. * Eric J Kremer Contributions Y.B. and M.B. planned and supervised the project and wrote the paper. Y.B. designed and performed most of the experiments with the help of C.C.-B. R.T. initiated the project. D.G. performed experiment in O.V.'s laboratory. C.G., C.D. and E.J.K. helped perform experiments and provided valuable reagents. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Yamina Bennasser or * Monsef Benkirane Author Details * Yamina Bennasser Contact Yamina Bennasser Search for this author in: * NPG journals * PubMed * Google Scholar * Christine Chable-Bessia Search for this author in: * NPG journals * PubMed * Google Scholar * Robinson Triboulet Search for this author in: * NPG journals * PubMed * Google Scholar * Derrick Gibbings Search for this author in: * NPG journals * PubMed * Google Scholar * Carole Gwizdek Search for this author in: * NPG journals * PubMed * Google Scholar * Catherine Dargemont Search for this author in: * NPG journals * PubMed * Google Scholar * Eric J Kremer Search for this author in: * NPG journals * PubMed * Google Scholar * Olivier Voinnet Search for this author in: * NPG journals * PubMed * Google Scholar * Monsef Benkirane Contact Monsef Benkirane Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (7M) Supplementary Figures 1–4 Additional data
• Critical nucleus size for disease-related polyglutamine aggregation is repeat-length dependent
  - Nat Struct Mol Biol 18(3):328-336 (2011)
  Nature Structural & Molecular Biology | Article Critical nucleus size for disease-related polyglutamine aggregation is repeat-length dependent * Karunakar Kar1, 2 * Murali Jayaraman1, 2 * Bankanidhi Sahoo1, 2 * Ravindra Kodali1, 2 * Ronald Wetzel1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:328–336Year published:(2011)DOI:doi:10.1038/nsmb.1992Received01 June 2010Accepted24 November 2010Published online13 February 2011 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 Because polyglutamine (polyQ) aggregate formation has been implicated as playing an important role in expanded CAG repeat diseases, it is important to understand the biophysics underlying the initiation of aggregation. Previously, we showed that relatively long polyQ peptides aggregate by nucleated growth polymerization and a monomeric critical nucleus. We show here that over a short range of repeat lengths, from Q23 to Q26, the size of the critical nucleus for aggregation increases from monomeric to dimeric to tetrameric. This variation in nucleus size suggests a common duplex antiparallel β-sheet framework for the nucleus, and it further supports the feasibility of an organized monomeric aggregation nucleus for longer polyQ repeat peptides. The data also suggest that a change in the size of aggregation nuclei may have a role in the pathogenicity of polyQ expansion in this series of familial neurodegenerative diseases. View full text Figures at a glance * Figure 1: Effect of AT7NT on polyQ aggregation kinetics. () PONDR analysis of the first 600 amino acids of the human HTT and AT7 sequences. Scores between 0.5 and 1 are associated with disorder and scores between 0 and 0.5 with order. The orange bars represent the polyQ sequence; the purple bars the proline-rich sequence. () Comparison of aggregation kinetics of peptide AT7NTQ30K2 and K2Q30K2. () Aggregation kinetics of different concentrations of AT7NTQ30K2. () Initial aggregation kinetics data of AT7NTQ30K2 from plotted against time2 (s2). () Log-log plots of initial reaction rates (from panel ) versus initial concentration for AT7NTQ30K2. () Autocorrelation functions obtained from DLS measurements at different incubation times of the aggregation reaction of AT7NTQ30K2 at 166 μM, along with a histogram of the percent aggregation at the same time points derived from HPLC-sedimentation analysis. * Figure 2: Transmission electron micrograph images obtained for different peptides incubated in PBS buffer at 37°C. (–) End-stage aggregates of AT7NTQ30K2 (), K2Q37K2 (), HTTNTQ37P10K2 () and SFQ37P10K2 (). (–) Representative images from different incubation times of a 215 μM reaction of K2Q23K2 observed at 0 h (), 24 h (,), 27 h (), 36 h (), 52 h () and 200 h (). The vast proportion of the EM grid examined for the 24-h time point for K2Q23K2 aggregation resembled (no visible aggregate). (–) End-stage aggregates of K2Q18K2 (), K2Q24K2 (), K2Q25K2 (), K2Q26K2 () and K2Q27K2 (). Scale bars, 50 nm. * Figure 3: Aggregation kinetics analysis of the peptide SFQ37P10K2. () Comparison of aggregation kinetics of HTTNTQ37P10K2, SFQ37P10K2 and K2Q37K2. () Aggregation kinetics for SFQ37P10K2 at different concentrations. () Initial aggregation kinetics data of SFQ37P10K2 from part b plotted against time2. () Log-log plot of initial reaction rates (slopes from ) versus initial concentration for SFQ37P10K2. * Figure 4: Aggregation kinetics of peptide K2Q37K2 in PBS at 37°C. () DLS measurements of the aggregation reaction mixture of K2Q37K2 at 25.5 μM starting concentration at the times indicated. () Aggregation kinetics for K2Q37K2 at different concentrations. () Initial aggregation kinetics data of K2Q37K2 from plotted against time2 (s2). () Log-log plots of initial reaction rates (slopes from ) versus initial concentration for K2Q37K2. * Figure 5: DLS analysis of the aggregation reaction of K2Q23K2 at 215 μM. () Autocorrelation functions obtained on freshly disaggregated and filtered 215-μM solution of K2Q23K2 (0 h) and after 2-h incubation at 37 °C in PBS. () Autocorrelation functions on the aggregation reaction of the 2-h reaction from , after first being subjected to a second 20-nm filtration (even though there is no indication of aggregate formation by DLS or by material loss on filtration; see Results). Also shown for this 215-μM reaction is a histogram of the percent aggregation at the same time points derived from HPLC-sedimentation analysis. () Hydrodynamic radius (Rh) derived from the DLS data in at t = 27 h. () The DLS data obtained for IgM at different concentrations measured in PBS at 37 °C. * Figure 6: Kinetic analysis of K2Q23K2 by sedimentation assay. () Aggregation kinetics of K2Q23K2 at different concentrations. The reaction labeled '103 μM + S' (blue, right-pointing triangles) contains ~12% by weight of K2Q23K2 aggregate seeds. () Time course of spontaneous fibril growth at 215 μM K2Q23K2 monomer, and of the dissociation of these fibrils, at 37 °C in PBS. The blue dashed line shows the approximate convergence point at about 3 μM, indicating a position of dynamic equilibrium of the forward and reverse reactions. () Initial aggregation kinetics data of K2Q23K2 from plotted against time2 (s2). () Log-log plots of initial reaction rates (slopes from ) versus initial concentration for K2Q23K2. * Figure 7: Log-log plots of initial reaction rates versus initial concentration. Rate data are from time2 plots (see Supplementary Fig. 2). (–) Peptides analyzed. K2Q18K2 (), K2Q27K2 (), K2Q26K2 (), K2Q25K2 () and K2Q24K2 (). * Figure 8: Further nucleation kinetics analysis of K2Q23K2. () Determination of pseudo-first-order elongation rate constant (k*) from elongation kinetics of K2Q23K2 seeded with ~12% (w/w) aggregates. () Determination of the femtomoles of biotinylated K2Q29K2 bound at 25 °C to aliquots of the K2Q23K2 seed fibril suspension (from panel ) to determine the concentration of growth sites in the seed aggregates (see Online Methods). () Linear fits of the log-log plot data of K2Q23K2 and K2Q37K2 (data from Figs. 6d and 4d, respectively), extrapolated to 1 nM. () Calculated aggregation kinetics curves for 1 nM of various polyQ peptides using parameters derived from nucleation kinetics analyses. * Figure 9: Models of nucleated growth polymerization. () Classical thermodynamic model of nucleation, where n* is the critical nucleus. (,) Two models for monomeric nucleation of polyQ aggregation in which the nucleus is either highly organized () or extended (). (–) Hypothetical 4-stranded β-sheet model structures for aggregation critical nuclei that are tetrameric (), monomeric () or dimeric (). Author information * Abstract * Author information * Supplementary information Affiliations * Structural Biology Department, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. * Karunakar Kar, * Murali Jayaraman, * Bankanidhi Sahoo, * Ravindra Kodali & * Ronald Wetzel * Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. * Karunakar Kar, * Murali Jayaraman, * Bankanidhi Sahoo, * Ravindra Kodali & * Ronald Wetzel Contributions K.K., M.J. and B.S. purified the peptides, determined and analyzed the aggregation kinetics and obtained dynamic light-scattering data on aggregation time points. R.K. obtained the EM and FTIR data. R.W. wrote the paper. All authors contributed to study design, data interpretation and improving the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ronald Wetzel Author Details * Karunakar Kar Search for this author in: * NPG journals * PubMed * Google Scholar * Murali Jayaraman Search for this author in: * NPG journals * PubMed * Google Scholar * Bankanidhi Sahoo Search for this author in: * NPG journals * PubMed * Google Scholar * Ravindra Kodali Search for this author in: * NPG journals * PubMed * Google Scholar * Ronald Wetzel Contact Ronald Wetzel Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (568K) Supplementary Figures 1–4 and Supplementary Table 1 Additional data
• Histone H3 lysine 9 trimethylation and HP1γ favor inclusion of alternative exons
  - Nat Struct Mol Biol 18(3):337-344 (2011)
  Nature Structural & Molecular Biology | Article Histone H3 lysine 9 trimethylation and HP1γ favor inclusion of alternative exons * Violaine Saint-André1, 2, 3 * Eric Batsché1, 2, 3 * Christophe Rachez1, 2, 3 * Christian Muchardt1, 2, 3 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:337–344Year published:(2011)DOI:doi:10.1038/nsmb.1995Received25 February 2010Accepted02 December 2010Published online27 February 2011 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 Pre-messenger RNAs (pre-mRNAs) maturation is initiated cotranscriptionally. It is therefore conceivable that chromatin-borne information participates in alternative splicing. Here we find that elevated levels of trimethylation of histone H3 on Lys9 (H3K9me3) are a characteristic of the alternative exons of several genes including CD44. On this gene the chromodomain protein HP1γ, frequently defined as a transcriptional repressor, facilitates inclusion of the alternative exons via a mechanism involving decreased RNA polymerase II elongation rate. In addition, accumulation of HP1γ on the variant region of the CD44 gene stabilizes association of the pre-mRNA with the chromatin. Altogether, our data provide evidence for localized histone modifications impacting alternative splicing. They further implicate HP1γ as a possible bridging molecule between the chromatin and the maturating mRNA, with a general impact on splicing decisions. View full text Figures at a glance * Figure 1: Histone H3K9 trimethylation (H3K9me3) is enriched on the CD44 alternative exons. (,) ChIP assays with antibodies to H3, H3K4me2, H3K4me3 and H3K36me3 () and antibodies to H3K9me2 and H3K9me3 (), and chromatin from HeLa cells. The relative enrichment of each modification along the CD44 gene was measured by qPCR using primer sets targeting the indicated exons and introns and is expressed as a fraction of the signal obtained with antibody to H3. The signal obtained upon immunoprecipitation with nonimmune IgG indicates the level of background (dotted line). Values are means ± s.e.m. of six qPCR values from one representative experiment of five independent experiments. Constant and variant exons are represented as black and gray boxes, respectively, on the CD44 gene map below the graphs. The proximal promoter (PP) corresponds to the transcriptional start site. * Figure 2: HP1γ regulates alternative splicing of the CD44 transcript. () HeLa cells were transfected with siHP1α, siHP1β, siHP1γ or siGAPDH as a control. Western blots on nuclear extracts were carried out with antibodies to HP1α (anti-HP1α), anti-HP1β or anti-HP1γ (lower panels) or anti-H3 (H3 used as a loading control, upper panels). Blots are representative of the experimental replicates. (,) RNAs from transfected cells were quantified by RT-qPCR. () Expression level of the CD44 C2C3 constant exons relative to that of RLP0 (set to 1) for each transfection condition. () Exon inclusion is measured as the ratio between the indicated CD44 exon couples and the constant exon couple C2C3 and expressed relatively to the values obtained upon transfection of siGAPDH (set to 1). Values are means ± s.e.m. of six qPCR values from three experimental replicates. () Western blots on nuclear extracts from HeLa S3 and HeLa S3-overexpressing HP1γ-HA (HeLa S3-HP1γ) cells were carried out with anti-HP1γ or anti-H3 (H3 used as a loading control). Blots! are representatives of the replicates. () RNAs from the corresponding HeLa S3 or HeLa S3-HP1γ cells were quantified by RT-qPCR. Exon inclusion is measured as the ratio between the indicated CD44 exon couples and the constant exon couple C2C3, and expressed relatively to the values obtained with the HeLa S3 cell line (set to 1). Values are means ± s.e.m. from four experimental replicates. * Figure 3: HP1γ, RNAP II and U2AF65 accumulate on the variant region of CD44 chromatin upon PKC stimulation. () HeLa cells were incubated (+PMA) or not (−PMA) overnight with PMA. After extraction of cytoplasmic RNA and DNase treatment, each CD44 exon was quantified by RT-qPCR. Exon inclusion is represented as fold change ± s.e.m. between stimulated and nonstimulated cells, with the signal from nonstimulated samples being set to 1. (–) ChIP walking experiments were carried out with antibodies to H3K9me3 (), HP1γ (), HP1γS83p (), U2AF65 () and RNAP II (8WG16) () and chromatin from HeLa cells treated (black bars and squares) or not (white bars and squares) for 2 h with PMA. Amounts of H3K9me3 are expressed in percent of H3, and amounts of HP1γ, U2AF65, RNAP II and HP1γS83p are expressed relatively to the signal obtained for ChIP using nonimmune IgG. Values are means ± s.e.m. of three independent experiments. () HeLa cells were stimulated with PMA for 2 h. Western blots on total protein extracts were carried out with the indicated antibodies. PP, proximal promoter. * Figure 4: HP1γ bridges chromatin to pre-mRNA. () Quantification by qPCR of CD44 pre-mRNA exons retained on chromatin. Non-cross-linked DNase-treated chromatin from HeLa cells stimulated (+PMA) or not (−PMA) with PMA for 2 h was extracted as described in Online Methods. Graph shows means ± s.e.m. of chromatin-bound RNA (quantified by RT-qPCR) relative to the DNA in the input (quantified by qPCR). (,) Quantification of CD44 pre-mRNA exons associated with HP1γ () and H3 () in the non-cross-linked chromatin fraction. RNA-ChIP assays were carried out with the indicated antibodies and chromatin prepared as in . Levels of immunoprecipitated RNA are expressed relatively to the levels of corresponding DNA sequences present in the extracted chromatin fraction. () Association of histone H3 with the CD44 pre-mRNA requires HP1γ. RNA-ChIPs were carried out with anti-H3 and cross-linked chromatin from HeLa cells transfected with the indicated siRNAs and stimulated with PMA for 2 h. The qPCR values are from a representative experi! ment and are expressed as a percent of input for each indicated amplicon. For all panels, values are means ± s.e.m. from two independent experiments. * Figure 5: Cell type–specific distribution of H3K9me3 and HP1γ correlates with variant exon inclusion and RNAP II accumulation. () Each CD44 exon was quantified by RT-qPCR after DNase treatment of SKOV3 (white) or SW626 (black) RNAs. Relative abundance of each exon on the mature transcript was measured by using the same external reference for each amplicon. Values are means ± s.e.m. from three independent experiments. (,) ChIP assays with antibodies to H3K9me3, H3 and HP1γ and chromatin from SW626 or SKOV3 cells. The relative enrichment on IL8 and GAPDH promoters, on satellite sequences (hSat) or along the CD44 gene was quantified by qPCR using primer sets targeting the exons and introns indicated on the map of the gene and expressed as a fraction of histone H3 in or relatively to IgG in (set to 1 for each cell line). Values are means ± s.e.m. of three independent qPCRs from one representative experiment. (,) Western blots with the indicated antibodies and nuclear protein extracts from SW626 or SKOV3 () or from SKOV3 and SKOV3 stably overexpressing HA-tagged HP1γ (SKOV3-HP1γ) (). () RT-qPCR quan! tification of RNAs from SKOV3 and SKOV3-HP1γ cells stimulated (+PMA) or not (−PMA) overnight with PMA. Exon inclusion is measured as the ratio between the variant v9v10 and the C16C17 exon couples. Values are means ± s.e.m. of three RT-qPCR from one representative experiment. (–) ChIP assays with antibodies to RNAP II (N20) (), HP1γ () and H3K9me3 and H3 (), and chromatin from SKOV3 or SKOV3-HP1γ transfected for 48 h with either siHP1γ or siGAPDH, then stimulated (+PMA) or not (−PMA) with PMA for 2 h. The relative enrichment along the CD44 gene was measured by qPCR and expressed in percent of H3 (for H3K9me3) or relatively to values obtained with IgG (for RNAP II and HP1γ). PP, proximal promoter. * Figure 6: HP1γ-dependent exon inclusion is guided by an H3K9me3 mark on several genes. HeLa cells were transfected with siHP1γ (black) or siGAPDH (gray) used as control. RNAs from the transfected cells were quantified by RT-qPCR using primer sets designed to amplify exon couples of which one exon (in black) is identified on GeneChip Human Exon 1.0 ST Arrays as less included upon depletion of HP1γ. (–) H3K9me3 ChIP (,, top). Panels show amounts of H3K9me3 expressed as a fraction of H3 estimated by ChIP at the indicated position. Transcripts are shown in ,, bottom, and –. Panels show the relative RNA amounts at the indicated position. Schematic of the mRNA represents to scale the coding exons of the longest isoform of the mRNA and the regulated splicing event for the indicated genes. Values were normalized to those for control genes HPRT and RLP0, whose expression is not affected by HP1γ depletion, and are means ± s.e.m. of three experimental replicates. * Figure 7: Model for the regulation of CD44 alternative splicing by H3K9me3 and HP1γ. () In the absence of the H3K9me3 mark, HP1γ is present at low levels on the chromatin, possibly via its interactions with the RNAP II or the globular domain of histone H3. Under these conditions the RNAP II elongates at high rates through the variant region of the gene, and the spliceosome recognizes only the splice sites of constant exons. These constant exons remain in contact with the chromatin via the spliceosome and the RNAP II, whereas the variant exons not bound by spliceosomes keep contact with the chromatin only via the RNAP II. Altogether, this results in inclusion of only the constant exons in the mature mRNA. () When levels of H3K9me3 increase inside the CD44 gene and/or when HP1γ becomes more available (possibly released from the heterochromatin by phosphorylation), HP1γ forms an additional link with chromatin via its chromodomain. It accumulates on the coding region where it associates with the pre-mRNA and favors its transient retention on the chromatin. Th! e generated chromatin structures slow down the RNAP II, which in turn facilitates recruitment of splicing factors such as U2AF65 and PRP8 on the pre-mRNA. This leads to increased inclusion of CD44 variant exons. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE25282 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Institut Pasteur, Département de Biologie du Développement, Unité de Régulation Epigénétique, Paris, France. * Violaine Saint-André, * Eric Batsché, * Christophe Rachez & * Christian Muchardt * Centre National de la Recherche Scientifique (CNRS), URA2578, Paris, France. * Violaine Saint-André, * Eric Batsché, * Christophe Rachez & * Christian Muchardt * INSERM Avenir, Paris, France. * Violaine Saint-André, * Eric Batsché, * Christophe Rachez & * Christian Muchardt Contributions V.S.-A., E.B. and C.R. designed, performed and analyzed the experiments and prepared the manuscript. C.M. conceived the project and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Christian Muchardt Author Details * Violaine Saint-André Search for this author in: * NPG journals * PubMed * Google Scholar * Eric Batsché Search for this author in: * NPG journals * PubMed * Google Scholar * Christophe Rachez Search for this author in: * NPG journals * PubMed * Google Scholar * Christian Muchardt Contact Christian Muchardt 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 (556K) Supplementary Figures 1–6 and Supplementary Tables 1 and 2 Additional data
• Mechanics of Hsp70 chaperones enables differential interaction with client proteins
  - Nat Struct Mol Biol 18(3):345-351 (2011)
  Nature Structural & Molecular Biology | Article Mechanics of Hsp70 chaperones enables differential interaction with client proteins * Rainer Schlecht1, 3 * Annette H Erbse1, 2, 3 * Bernd Bukau1 * Matthias P Mayer1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:345–351Year published:(2011)DOI:doi:10.1038/nsmb.2006Received14 July 2010Accepted14 December 2010Published online30 January 2011 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 Hsp70 chaperones interact with a wide spectrum of substrates ranging from unfolded to natively folded and aggregated proteins. Structural evidence suggests that bound substrates are entirely enclosed in a β-sheet cavity covered by a helical lid, which requires structural rearrangements including lid opening to allow substrate access. We analyzed the mechanics of the lid movement of bacterial DnaK by disulfide fixation of lid elements to the β-sheet and by electron paramagnetic resonance spectroscopy using spin labels in the lid and β-sheet. Our results indicate that the lid-forming helix B adopts at least three conformational states and, notably, does not close over bound proteins, implying that DnaK does not only bind to extended peptide stretches of protein substrates but can also accommodate regions with substantial tertiary structure. This flexible binding mechanism provides a basis for the broad spectrum of substrate conformers of Hsp70s. View full text Figures at a glance * Figure 1: Structure of the DnaK substrate-binding domain. () Illustration of E444C, A519C, K446C, D526C, E430R1 and R547R1′ alterations in the crystal structure of the SBD of DnaK (PDB 1DKX10) in cartoon representation. The β-sheet subdomain is drawn in cyan, the helical lid in blue and the cocrystallized substrate peptide NRLLTG in red. Modified residues are indicated. The E430R1,R547R1′ side chain pair in energy-minimized conformations are shown as stick models to illustrate the expected close proximity of the spin-labeled side chains in the 'closed lid' conformation. At right is the structure of the spin-labeled side chains illustrating the range (<20 Å) of detectable interactions33. Structural representations were created in PyMOL (http://www.pymol.org/). () A space-filling representation of the SBD shown in . () Models of the proposed lid opening mechanisms: (left to right) (i) melting of helix B opposite the substrate-binding cavity and subsequent kinking of the C-terminal helical bundle; (ii) upward rotation of helix B! around a hinge between helix A and helix B; (iii) sideward movement of the lid by pivoting around helix A; and (iv) formation of a continuous helix A and B and complete detachment of the helical subdomain from the β-sheet domain, similar to the Hsp110 structure. * Figure 2: DnaK variants show altered interaction with substrates. () Peptide substrates dissociated fastest from reduced DnaK-HB. Shown are unstimulated dissociation rate constants obtained for the dissociation of DNR peptide from oxidized or reduced DnaK wild-type or double cysteine variants (please note the break in the y axis). Fluorescence decay was fitted by a single exponential equation. () The reduced form of DnaK-HB showed the fastest association rate. Shown are apparent peptide association rates observed for the interaction of 0.5 μM DnaK with 2 μM DNR-peptide in a stopped-flow device. Rates for the fast (gray) and slow (white) phase and the amplitude of the fast phase (% fast) are given. Error bars represent the standard error of three independent experiments. () Elution profiles of gel filtration analysis over a Superdex 200 10/30 column (GE Healthcare). We incubated different concentrations of oxidized DnaK-HB (0–50 μM) with 3H-σ32 (1 μM, 1,500 Bq) and separated the bound and unbound fractions by gel filtration. The amou! nt of radio-labeled substrate protein is plotted against the elution volume. Peak I corresponds to the DnaK-σ32 complex and peak II corresponds to free transcription factor. c.p.m., counts per minute. () Substrate affinity is not altered in DnaK double-cysteine mutants. Dissociation equilibrium titration of 3H-labeled σ32 with 0–50 μM oxidized wild-type DnaK and double-cysteine variants. We determined the Kd values by fitting the quadratic solution to the data (Table 1). Ox, oxidized. * Figure 3: Only reduced DnaK-HA is capable of refolding luciferase. (–) Refolding of chemically denatured firefly luciferase by wild-type DnaK and double-cysteine mutants under reducing () or oxidizing () conditions (80 nM luciferase, 800 nM DnaK, 160 nM DnaJ, 400 nM GrpE and 20 mM DTT for ). Luciferase activity is plotted as a fraction of the native luciferase control. Neg. control, negative control. * Figure 4: Effects of disulfide bond formation on interdomain communication. () Restricting lid mobility alters basal and stimulated ATPase activity of DnaK. Single-turnover ATPase rates in the absence and presence of 50 nM DnaJ and 1 μM σ32 under oxidizing (gray) and reducing conditions (white). () Oxidized DnaK-HA is impaired in ATP-stimulated peptide release. Peptide dissociation rates in the presence of ATP are shown. For reduced DnaK-HA, we observed a second phase (white bar). () Tryptophan fluorescence revealed defects in ATP-induced conformational changes in oxidized double-cysteine mutants. Difference of the wavelength of the emission maximum of tryptophan fluorescence in the presence of ATP minus the wavelength of the maximum in the absence of nucleotide. Reduced samples contain 20 mM DTT. Error bars represent the standard error of three independent experiments. Ox, oxidized; red, reduced. * Figure 5: EPR spectra of a spin pair in the SBD of DnaK in the presence of nucleotide and substrate. () Spectra of DnaK E430R1,R547R1′ (50 μM) in the absence (left) and presence (right) of ATP (2 mM) are shown. Comparison of the experimental spectrum of the double mutant (red line) with the algebraic sum of the spectra of the corresponding single mutants, normalized to the same number of spins (gray trace). () Derived interspin distance distribution between the spin pair in the absence and presence of ATP from fits to the spectra in (Supplementary Fig. 6b). The y axis is arbitrary and chosen for the ease of presentation. The population of non-interacting spin pairs (distance > 20 Å) is not shown, but the fraction of total spin contributed to this population (fNI) is given. () Investigation of the spin-spin interaction in the presence of the folded protein substrate σ32 (300 μM). The left panel shows the comparison of the experimental spectrum of the double mutant (blue line) with the algebraic sum of the spectra of the corresponding single mutants (gray line) in the p! resence of bound protein, normalized to the same number of spins. The right panel shows an overlay of the experimental EPR spectrum of the double mutant in the presence of ATP (red line) with the spectrum of the mutant in the presence of σ32 (blue line), illustrating that the spectrum of protein-substrate–bound DnaK is similar to the ATP bound, 'open lid' conformation. * Figure 6: The distal part of helix B can be cross-linked to bound substrate. We labeled DnaK-Q424C and DnaK-R547C with the heterobifunctional, thiol-specific, UV-inducible cross-linker 4-(2-Iodoacetamido)benzophenone (BPIA) and cross-linked to yellow fluorescent protein (YFP) and σ32 by irradiation with UV light39. We visualized His-tagged substrates and cross-linking products by immunoblotting with a Penta-His Antibody (QIAGEN). Bands corresponding to DnaK-substrate complexes can be detected when BPIA is linked either to the β-sheet subdomain close to the substrate binding pocket (Q424C, compare to ref. 39) or to the distal part of helix B (R547C), however this is true only for the native substrate protein σ32 and not for YFP, which is not bound by DnaK in its native state. MW, molecular weight. * Figure 7: Cartoon representation of the SBD of DnaK illustrating possible lid movements. () The 'closed lid' crystal structure is depicted as a dark gray cartoon and the R1 side chains as a dark gray stick model. The green translucent helical lid illustrates a distance of the nitroxide groups of the R1 moieties of 18 Å with a kinking of helix B opposite the substrate-binding pocket. The blue translucent lid visualizes a possible wide-open structure with a distance of 38 Å between the R1 groups. Because a distance of 38 Å is far beyond the detection range of dipolar spin-spin interactions with the method used, it remains unclear whether the lid actually opens to this extent. () Multiple functions of Hsp70 chaperones. Some are likely to allow lid closure, whereas others are rather unlikely to do so. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 1DKX * 1DKX Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Rainer Schlecht & * Annette H Erbse Affiliations * Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ–ZMBH Allianz, Im Neuenheimer Feld 282, Heidelberg, Germany. * Rainer Schlecht, * Annette H Erbse, * Bernd Bukau & * Matthias P Mayer * Present address: Department of Chemistry and Biochemistry, University of Colorado Boulder, Boulder, Colorado, USA. * Annette H Erbse Contributions R.S. designed, performed and interpreted the experiments in Figures 2,3,4 and Figure 6, Table 1, Supplementary Figures 1,4,5 and 9 and Supplementary Table 1. A.H.E. designed, performed and interpreted the experiments in Figure 5 and Supplementary Figures 6–8. B.B. designed the experiments and supervised R.S. and A.H.E. M.P.M. designed all mutant proteins; designed, performed and interpreted experiments in Supplementary Figure 2; was involved in the design and interpretation of all experiments; and supervised R.S. and A.H.E. All authors prepared figures and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Matthias P Mayer or * Bernd Bukau Author Details * Rainer Schlecht Search for this author in: * NPG journals * PubMed * Google Scholar * Annette H Erbse Search for this author in: * NPG journals * PubMed * Google Scholar * Bernd Bukau Contact Bernd Bukau Search for this author in: * NPG journals * PubMed * Google Scholar * Matthias P Mayer Contact Matthias P Mayer 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 Table 1 Additional data
• Structure-function analysis of hRPC62 provides insights into RNA polymerase III transcription initiation
  - Nat Struct Mol Biol 18(3):352-358 (2011)
  Nature Structural & Molecular Biology | Article Structure-function analysis of hRPC62 provides insights into RNA polymerase III transcription initiation * Stéphane Lefèvre1, 2 * Hélène Dumay-Odelot1, 2 * Leyla El-Ayoubi1, 2 * Aidan Budd3 * Pierre Legrand4 * Noël Pinaud1, 2, 5 * Martin Teichmann1, 2 * Sébastien Fribourg1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:352–358Year published:(2011)DOI:doi:10.1038/nsmb.1996Received18 June 2010Accepted02 December 2010Published online27 February 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The 17-subunit human RNA polymerase III (hPol III) transcribes small, untranslated RNA genes that are involved in the regulation of transcription, splicing and translation. hPol III subunits hRPC62, hRPC39 and hRPC32 form a stable ternary subcomplex required for promoter-specific transcription initiation by hPol III. Here, we report the crystal structure of hRPC62. This subunit folds as a four-tandem extended winged helix (eWH) protein that is structurally related to the transcription factor TFIIEα N terminus. Through biochemical analyses, we mapped the protein-protein interactions of hRPC62, hRPC32 and hRPC39. In addition, we demonstrated that hRPC62 and hRPC39 bind single-stranded and duplex DNA, respectively, in a sequence-independent manner. Overall, we shed light on structural similarities between the hPol III–specific subunit hRPC62 and TFIIEα and propose specific functions for hRPC39 and hRPC62 in transcription initiation by hPol III. View full text Figures at a glance * Figure 1: Overall structure of hRPC62. () Structure of hRPC62 is represented as a ribbon diagram. The four extended winged helix domains (eWH) are shown in red (eWH1), green (eWH2), blue (eWH3) and orange (eWH4). The four eWH domains form a globular head, whereas the two C-terminal long α helices protruding out of the protein form the C-terminal tail. Dotted lines represent undefined protein segments and are labeled with the amino acid number at that position. The two orientations are related by a horizontal 90° rotation. () Structural similarity between eWH1, eWH3 and TFE, the archaeal homolog of TFIIEα. A 3-D superimposition of eWH1 (red), eWH3 (blue) and TFE (gray, PDB code 1Q1H)21 is shown. The r.m.s. deviation between the C-α backbone of eWH1 and eWH3 and TFE is given in parentheses. () Domain organization of hRPC62. The first eWH1 encompasses residues 1–78; eWH2, 88–237; eWH3, 239–337; eWH4, 338–426; and the coiled-coil domain, 428–457 and 493–534. * Figure 2: Interaction mapping of the two isoforms of hRPC32 with hRPC62. The hRPC62 eWH3 is necessary for the interaction with hRPC32 paralogs, whereas the hRPC62 coiled-coil domain is needed for interaction with hRPC32β and not with hRPC32α. The two-hybrid system was used to monitor protein-protein interactions between hRPC62 and hRPC32α or hRPC32β. Control experiments are shown in the central panel, whereas assays with hRPC32α and hRPC32β are shown respectively on the left and right sides. Gray bars stand for the control experiment with an empty plasmid (GAD); red and blue bars correspond respectively to hRPC32α and hRPC32β tests. Deletion constructs of hRPC62 were tested as well as point mutants. Values represent the average of three independent experiments, and error bars indicate s.d. * Figure 3: Mutual-interaction mapping of hRPC39 and hRPC62. () Pulldown experiments of hRPC62 deletion constructs with hRPC39. The eWH domains of hRPC62 are required to bind hRPC39, whereas the coiled-coil domain and the eWH2 insertion loop are not. () hRPC62 point mutants' interaction with hRPC39. Point mutants spanning hRPC62 domains do not alter the hRPC39 interaction. () Pulldown experiments of hRPC39 deletion constructs with hRPC62. The last 158 residues of hRPC39 are sufficient to bind hRPC62, whereas the two first WH domains are dispensable for interaction. () hRPC39 point-mutant pulldowns. Equivalent point mutations originally identified in the Rpc34 subunit do not alter the hRPC39-hRPC62 interaction9. * Figure 4: DNA-binding properties of hRPC62. () Single- versus double-stranded–DNA–binding properties of hRPC62. Left, wild-type hRPC62 binds ssDNA but not dsDNA in a sequence-independent manner. Right, hRPC62 ssDNA binding is specific and independent of the DNA sequence. In a competition assay, a scrambled oligonucleotide of the same length is able to displace hRPC62 from its original target, whereas dsDNA probe does not compete with ssDNA. () Surface charge and conservation of hRPC62. Red and blue indicate negative and positive charges, respectively (left panel). Several conserved residues tested for their DNA-binding properties are shown and labeled. Ribbon diagram and surface representation of hRPC62 are shown in the same orientation as in the middle panel. The sequence conservation at the surface of the hRPC62 is displayed from white (nonconserved) to red (highly conserved) in the right panel. () DNA-binding properties of hRPC62 point mutants and deletion constructs. Alteration of ssDNA binding is observed for! point and deletion mutants targeting conserved residues at the surface of hRPC62 domains. * Figure 5: DNA-binding properties of hRPC39. () Single- versus double-stranded–DNA–binding properties of hRPC39. hRPC39 binds strongly to dsDNA and mildly to ssDNA (left). Incubation with an antibody directed against the His tag of hRPC39 recombinant protein shifts the hRPC39-ssDNA retardation band and proves the interaction's specificity. () ssDNA can be supershifted upon serial addition of hRPC62, hRPC39 and an antibody directed against hRPC62. () DNA-binding properties of hRPC39 point mutants and deletion constructs. The first WH domain of hRPC39 is not needed for dsDNA recognition. The human equivalent rpc34-1109 (K137E–K140E) and rpc34-1124 (E173K–E175K) mutants are severely altered in dsDNA recognition. * Figure 6: hRPC62 (Rpc82) positioning into the yeast RNA Pol III electron-density map. () Graphical summary of reported interactions. hRPC62 domains follow the color coding used in the paper (eWH1 in red, eWH2 in green, eWH3 in blue, eWH4 in orange, coiled coil in pink). eWH3 is needed for hRPC32α/β binding to hRPC62, whereas the coiled-coil domain provides an additional binding surface to hRPC32β. All eWHs are needed for the interaction with hRPC39 and ssDNA. dsDNA binding by RPC39 requires WH2 and the C terminus, whereas the latter is sufficient for interacting with hRPC62. () hRPC62 (Rpc82) positioning in the yeast RNA Pol III electron density map. The yeast RNA Pol II–TFIIB complex (PDB code 3KIF, gray surface) was fitted into the EM envelope of yeast Pol III (blue mesh)31. hRPC62 was then placed in the empty density. The putative localization of hRPC39 is shown as a black dotted line. Brf1 is colored in purple. The two orientations are related by a 90° rotation in the plane axis. The equivalent position to the T69N point mutation is indicated52. Fre! e additional density (AD) 1 and 3 are labeled28. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 2XUB * 2XV4 * 2XUB * 2XV4 Referenced accessions Protein Data Bank * 2DK5 * 2DK8 * 1Q1H * 3KIF * 2DK5 * 2DK8 * 1Q1H * 3KIF Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Institut Européen de Chimie et Biologie, Pessac, France. * Stéphane Lefèvre, * Hélène Dumay-Odelot, * Leyla El-Ayoubi, * Noël Pinaud, * Martin Teichmann & * Sébastien Fribourg * Inserm and Université de Bordeaux, Bordeaux, France. * Stéphane Lefèvre, * Hélène Dumay-Odelot, * Leyla El-Ayoubi, * Noël Pinaud, * Martin Teichmann & * Sébastien Fribourg * European Molecular Biology Laboratory, Heidelberg, Germany. * Aidan Budd * Synchrotron SOLEIL, St-Aubin, France. * Pierre Legrand * CESAMO ISM, Université de Bordeaux, Pessac, France. * Noël Pinaud Contributions S.L. and N.P. purified and crystallized hRPC62. S.L., P.L. and S.F. determined the crystal structure of hRPC62. A.B. performed the bioinformatics analysis. H.D.-O., L.E.-A. and S.F. conducted functional assays. M.T. contributed to manuscript preparation. S.F. designed, supervised research and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Sébastien Fribourg Author Details * Stéphane Lefèvre Search for this author in: * NPG journals * PubMed * Google Scholar * Hélène Dumay-Odelot Search for this author in: * NPG journals * PubMed * Google Scholar * Leyla El-Ayoubi Search for this author in: * NPG journals * PubMed * Google Scholar * Aidan Budd Search for this author in: * NPG journals * PubMed * Google Scholar * Pierre Legrand Search for this author in: * NPG journals * PubMed * Google Scholar * Noël Pinaud Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Teichmann Search for this author in: * NPG journals * PubMed * Google Scholar * Sébastien Fribourg Contact Sébastien Fribourg Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–5 and Supplementary Methods Additional data
• The glmS riboswitch integrates signals from activating and inhibitory metabolites in vivo
  - Nat Struct Mol Biol 18(3):359-363 (2011)
  Nature Structural & Molecular Biology | Article The glmS riboswitch integrates signals from activating and inhibitory metabolites in vivo * Peter Y Watson1, 2, 3 * Martha J Fedor1, 2, 3 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:359–363Year published:(2011)DOI:doi:10.1038/nsmb.1989Received24 August 2010Accepted25 November 2010Published online13 February 2011 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 glmS riboswitch belongs to the family of regulatory RNAs that provide feedback regulation of metabolic genes. It is also a ribozyme that self-cleaves upon binding glucosamine-6-phosphate, the product of the enzyme encoded by glmS. The ligand concentration dependence of intracellular self-cleavage kinetics was measured for the first time in a yeast model system and unexpectedly revealed that this riboswitch is subject to inhibition as well as activation by hexose metabolites. Reporter gene experiments in Bacillus subtilis confirmed that this riboswitch integrates positive and negative chemical signals in its natural biological context. Contrary to the conventional view that a riboswitch responds to just a single cognate metabolite, our new model proposes that a single riboswitch integrates information from an array of chemical signals to modulate gene expression based on the overall metabolic state of the cell. View full text Figures at a glance * Figure 1: System for measuring intracellular glmS riboswitch cleavage kinetics. () The glmS riboswitch from Bacillus anthracis27. Cleavage at the site indicated by the arrow yields 5′ product (5′ P) and 3′ product (3′ P) RNAs. () The riboswitch coding sequence was inserted into the 3′ UTR of the yeast PGK1 gene and expressed under the control of a galactose-inducible promoter, the Gal Upstream Activation Sequence (UASGAL). Full-length glmS chimeric mRNA with a WT riboswitch decays faster than an mRNA with an inactivating G33A mutation10 and is less abundant at steady state owing to the contribution of cleavage to decay kinetics9. Comparison of chimeric mRNAs with a WT riboswitch to mRNA with the inactivating G33A mutation ensures that changes in decay kinetics reflect cleavage and not an effect of the sequence insertion on mRNA stability. Chimeric mRNA is shown capped (CAP) and polyadenylated (An). * Figure 2: GlcN dependence of glmS riboswitch cleavage in yeast. () RNase protection assays revealed full-length (336 nt) and 5′ P and 3′ P cleavage product RNAs (121 and 215 nt, respectively) in yeast expressing WT and G1A chimeric mRNAs during growth in medium with galactose and 10 mM GlcN along with RNA fragments upstream and downstream of the riboswitch insert in genomic PGK1 mRNA (93 and 70 nt, respectively). Only full-length mRNA was detected in yeast expressing the mutationally inactivated G33A riboswitch. ACT1 mRNA (191 nt) was used for normalization. () Lower abundance of full-length chimeric mRNAs with WT and G1A riboswitch insertions relative to chimeric mRNA with an inactivating G33A mutation during growth in galactose and different GlcN concentrations reflects GlcN-dependent cleavage. Bars represent the mean and error bars represent the s.d. of two or more experiments. FL, full-length. () Intracellular cleavage rates were calculated from the steady-state abundance9 of chimeric mRNAs with WT or G1A riboswitch inserts and f! it to the Michaelis-Menten equation to calculate apparent values. Points represent the mean and error bars represent the s.d. of rates calculated from the data shown in . () GlcN dependence of glmS riboswitch cleavage kinetics measured under conditions that approximate a physiological ionic environment in vitro. The plot shows rates measured in a representative experiment and error bars represent the standard error of the fit. Reported kmax and values represent the mean and s.d. of values obtained from three or more experiments. * Figure 3: Chimeric mRNA cleavage and decay kinetics after transcription inhibition in different carbon sources. () Full-length (FL) WT and G33A chimeric mRNA showed similar decay rates after transcription inhibition by transfer of yeast from galactose to glucose with 5 mM GlcN. Chimeric mRNA levels are shown normalized to the initial abundance of full-length riboswitch RNA (FL0). () WT chimeric mRNA showed faster decay than chimeric mRNA with an inactivating G33A mutation after transcription inhibition in glycerol, reflecting the contribution of riboswitch cleavage. Cleavage rates were calculated from the difference between the decay rate of full-length chimeric mRNA with a G33A riboswitch insert, which reflects degradation through the endogenous mRNA turnover pathway, and chimeric mRNA with a WT riboswitch insert, which decays through both cleavage and normal degradation pathways9. Addition of 5 mM GlcN after transcription inhibition accelerated decay of chimeric mRNA with a WT riboswitch even further. Decay kinetics for G33A mutant chimeric were the same under all conditions for whi! ch WT decay rates are reported, indicating that mRNA turnover through endogenous mRNA turnover pathways was not affected by riboswitch insertions or change of carbon source. Reported rates represent the mean and s.d. of values obtained from two or more replicate experiments. * Figure 4: Hexoses inhibit riboswitch cleavage in vitro and in Bacillus subtilis. () Cleavage rates were measured in 5mM GlcN and varying concentrations of Glc6P under conditions that approximate a physiological ionic environment in vitro. The plot shows rates measured in a representative experiment and error bars represent the standard error of the fit. The reported values represent the mean and s.d. of values obtained from two or more experiments. () Riboswitch activity in B. subtilis with a GFP-coding sequence fused to the WT (WTGFP) or inactive (G33AGFP) riboswitch. WTGFP showed a significant (*P < 0.001) reduction in relative fluorescence units (RFU) relative to G33AGFP during growth in minimal medium with GlcN, indicating that cleavage decreased GFP mRNA abundance, as expected. Fluorescence intensity increased significantly in bacteria with WTGFP (**P < 0.005) in medium with glucose and GlcN but did not change in bacteria with G33AGFP (P > 0.3), evidence that glucose or a glucose-derived metabolite prevented riboswitch cleavage from reducing WTGFP m! RNA abundance. Bars represent the mean and s.d. of fluorescence emission intensity obtained from six replicate experiments. Statistical significance was determined using a one-tailed Student's t-test. * Figure 5: Model for riboswitch regulation of GlmS gene expression. Riboswitch cleavage activity integrates information about the metabolic state of the cell by responding to the concentrations and affinities of an array of chemical signals. In this model, hexoses increase glmS mRNA abundance and upregulate GlmS expression by inhibiting riboswitch cleavage (green line), whereas aminohexoses decrease glmS mRNA abundance and downregulate GlmS expression by activating riboswitch cleavage (red arrow). The dashed box illustrates the former model that a single cognate ligand, GlcN6P, activates the riboswitch to downregulate GlmS expression. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA. * Peter Y Watson & * Martha J Fedor * Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA. * Peter Y Watson & * Martha J Fedor * The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, USA. * Peter Y Watson & * Martha J Fedor Contributions P.Y.W. and M.J.F. conceived of and designed the experiments, analyzed the data and wrote the paper. P.Y.W. conducted the experiments. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Martha J Fedor Author Details * Peter Y Watson Search for this author in: * NPG journals * PubMed * Google Scholar * Martha J Fedor Contact Martha J Fedor Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (139K) Supplementary Figure 1 and Supplementary Table 1 Additional data
• Defining an allosteric circuit in the cysteine protease domain of Clostridium difficile toxins
  - Nat Struct Mol Biol 18(3):364-371 (2011)
  Nature Structural & Molecular Biology | Article Defining an allosteric circuit in the cysteine protease domain of Clostridium difficile toxins * Aimee Shen1 * Patrick J Lupardus2, 3, 7 * Malte M Gersch4 * Aaron W Puri5 * Victoria E Albrow1, 7 * K Christopher Garcia2, 3 * Matthew Bogyo1, 6 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:364–371Year published:(2011)DOI:doi:10.1038/nsmb.1990Received29 September 2010Accepted24 November 2010Published online13 February 2011 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 An internal cysteine protease domain (CPD) autoproteolytically regulates Clostridium difficile glucosylating toxins by releasing a cytotoxic effector domain into target cells. CPD activity is itself allosterically regulated by the eukaryote-specific molecule inositol hexakisphosphate (InsP6). Although allostery controls the function of most proteins, the molecular details underlying this regulatory mechanism are often difficult to characterize. Here we use chemical probes to show that apo-CPD is in dynamic equilibrium between active and inactive states. InsP6 markedly shifts this equilibrium toward an active conformer that is further restrained upon binding a suicide substrate. Structural analyses combined with systematic mutational and disulfide bond engineering studies show that residues within a β-hairpin region functionally couple the InsP6-binding site to the active site. Collectively, our results identify an allosteric circuit that allows bacterial virulence factors t! o sense and respond to the eukaryotic environment. View full text Figures at a glance * Figure 1: Development of an activity-based probe assay for measuring TcdB CPD activity. () Structure of AWP19 activity-based probe. Residues in the peptide specificity element, AOMK electrophile and Cy5 fluorophore are marked. () InsP6-induced labeling of TcdB CPD by AWP19. Recombinant TcdB CPD was mixed with AWP19 and incubated with the indicated concentration of InsP6. The labeling reactions were resolved by SDS-PAGE, and fluorescence was measured using a flatbed scanner, followed by visualization of total protein using Coomassie blue staining. Wild type, WT. () Quantification of EC50 for the AWP19 labeling assay. AU, arbitrary units. * Figure 2: Detection of an active conformer in the apo state using an activity-based probe. () Time-dependent labeling of apo-CPD by AWP19. TcdB CPD, without exogenous InsP6, was incubated with AWP19 for indicated period of time, and reactions were resolved by SDS-PAGE. Probe labeling was visualized by fluorescent scanning. Percent labeling was compared relative to TcdB CPD incubated with InsP6 and AWP19 in parallel. () Preincubation of TcdB CPD with an AOMK inhibitor leads to time-dependent inhibition of TcdB CPD activity. TcdB CPD was incubated with Ac-GSL-AOMK with or without InsP6 for the indicated times. Residual activity was measured by labeling the enzyme with AWP19 in the presence of InsP6. () Rate of AWP19 labeling of TcdB CPD in response to increasing InsP6 concentrations. Fluorescent signal (arbitrary units, AU) versus time. () Rate of AWP19 labeling measured in versus InsP6 concentration. AWP19 labeling exhibited Michaelis-Menten–like kinetics, allowing for the determination of a dissociation constant of 0.4 μM. This value is in agreement with the pu! blished Kd determined using nonenzymatic methods5. * Figure 3: Effect of InsP6 and P1 leucine inhibitor modification on TcdB CPD conformational mobility. () Limited proteolysis profile of TcdB CPD variants with and without InsP6. TcdB CPD was untreated, treated with NEM or treated with Ac-GSL-AOMK for 16 h. TcdB CPD variants were repurified using the His6 tag and then subjected to limited proteolysis using chymotrypsin without (−) or with (+) InsP6. Reactions were resolved by SDS-PAGE and visualized by Coomassie blue staining. () Trypsin digestion of TcdB CPD exposed to InsP6 with or without covalent inhibitors. TcdB CPD was activated with InsP6 alone, or with InsP6 and the indicated inhibitor. Treated CPDs were repurified using gel filtration to remove inhibitor and excess InsP6. Untreated enzyme was also subjected to the same repurification procedure. Repurified TcdB CPD variants were digested with trypsin (0.2 mg ml−1), and aliquots were removed at indicated time points and resolved by SDS-PAGE followed by Coomassie blue staining. () InsP6 transfer assay. Repurified TcdB CPD variants from before addition of trypsin (in! put) and after trypsin digestion (trypsin) were added to MARTXVc CPD and fluorogenic substrate cleavage was measured. InsP6 was also added directly to the fluorogenic cleavage assay to determine maximal protease activation of MARTXVc CPD. Percentages above bars indicate amount of exogenous InsP6 transferred to MARTXVc CPD relative to total amount of InsP6 that could have been transferred assuming each TcdB CPD molecule carried a single InsP6 molecule through the repurification procedure. Values are mean ± s.d. (n = 3). * Figure 4: Structure of TcdB CPD and comparison with TcdA CPD. () Overview of TcdB CPD structure. Left, InsP6-binding site; right, structure rotated 120° to show the active site. The catalytic cysteine and histidine residues are stick models, and the β-flap and bound InsP6 are labeled. The overall structure is colored from blue at the N terminus to red at the C terminus for orientation. () Overlay of the TcdB (green) and TcdA (light blue) CPD InsP6-binding sites. Side chains within 3.5 Å of InsP6 are labeled (TcdB/TcdA) and shown as stick models. () Overlay of the TcdB (green) and TcdA (light blue) CPD active sites. Approximate S1 pocket that interacts with the P1 leucine is shaded. The catalytic cysteine and histidine residues are transparent spheres and in red lettering; conserved residues near the active site are stick models. Residue numbering is given as TcdA/TcdB. * Figure 5: InsP6 binding induces movement of conserved tryptophan in β-flap region. () Effect of InsP6 on intrinsic tryptophan fluorescence of TcdB CPD. TcdB CPD was incubated with the indicated concentration of InsP6, and the fluorescence emission spectra were measured after excitation at 295 nm. Relative fluorescence units were normalized to A280 values. () Close-up view of β-flap region. β-flap ribbon structure is purple, and residues required for CPD activity are magenta sticks. Select InsP6-interacting residues are cyan sticks. Residues dispensable for CPD activity are purple sticks. EC50 values for different point mutants were measured using the probe labeling assay and are mean ± s.d. (n = 3). A lower EC50 indicates that the mutant is more sensitive to InsP6. Wild type, WT. (,) Tryptophan fluorescence emission of wild-type and β-flap mutants (), and InsP6-binding mutants () at 340 nm was measured in the presence of increasing concentrations of InsP6. * Figure 6: Effect of redox state on InsP6 responsiveness of engineered disulfide bond mutants in the β-flap. The β-flap is a purple ribbon structure. Residues mutated to cysteine as pairs are sticks and colored similarly. Catalytic residues are red sticks. EC50 for each mutant is shown with (+DTT) or without (S–S) reductant. A lower EC50 indicates that the mutant is more sensitive to InsP6. Data are mean ± s.d. (n = 3). Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3PEE * 3PEE Referenced accessions Protein Data Bank * 3PA8 * 3PA8 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Pathology, Stanford School of Medicine, Stanford, California, USA. * Aimee Shen, * Victoria E Albrow & * Matthew Bogyo * Department of Molecular and Cellular Physiology, Stanford School of Medicine, Stanford, California, USA. * Patrick J Lupardus & * K Christopher Garcia * Howard Hughes Institute, Stanford School of Medicine, Stanford, California, USA. * Patrick J Lupardus & * K Christopher Garcia * Department of Chemical and Systems Biology, Stanford School of Medicine, Stanford, California, USA. * Malte M Gersch * Department of Chemistry, Center for Integrated Protein Science Munich (CIPSM), Technische Universitat Munchen, Garching, Germany. * Aaron W Puri * Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, California, USA. * Matthew Bogyo * Current addresses: Genentech, South San Francisco, California, USA (P.J.L.); Pfizer, Sandwich, UK (V.E.A.). * Patrick J Lupardus & * Victoria E Albrow Contributions A.S. developed and executed autocleavage, probe labeling, limited proteolysis and tryptophan fluorescence assays; constructed, purified and expressed CPD variants; prepared CPD for crystallization studies; helped design the AWP19 probe; and wrote the manuscript. P.J.L. crystallized InsP6-bound TcdB CPD, collected the data, solved and analyzed the structure, generated Figure 4 and Table 1 and provided advice on the manuscript. M.M.G. synthesized AWP19, helped with initial characterization of TcdB CPD mutants using a fluorogenic substrate assay and the probe labeling assay and provided creative input. A.W.P. helped design and synthesize the AWP19 probe and performed initial characterization of probe labeling of TcdB CPD. V.E.A. helped design the AWP19 probe and provided advice on probe usage. K.C.G. provided advice on the manuscript and provided financial support. M.B. provided creative input and financial support, and helped write the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Matthew Bogyo Author Details * Aimee Shen Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick J Lupardus Search for this author in: * NPG journals * PubMed * Google Scholar * Malte M Gersch Search for this author in: * NPG journals * PubMed * Google Scholar * Aaron W Puri Search for this author in: * NPG journals * PubMed * Google Scholar * Victoria E Albrow Search for this author in: * NPG journals * PubMed * Google Scholar * K Christopher Garcia Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew Bogyo Contact Matthew Bogyo 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 1–6, Supplementary Tables 1 and 2, and Supplementary Methods Additional data
• Mapping of INS promoter interactions reveals its role in long-range regulation of SYT8 transcription
  - Nat Struct Mol Biol 18(3):372-378 (2011)
  Nature Structural & Molecular Biology | Article Mapping of INS promoter interactions reveals its role in long-range regulation of SYT8 transcription * Zhixiong Xu1 * Gang Wei2 * Iouri Chepelev2 * Keji Zhao2 * Gary Felsenfeld1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:372–378Year published:(2011)DOI:doi:10.1038/nsmb.1993Received24 June 2010Accepted30 November 2010Published online20 February 2011 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 Insulin (INS) synthesis and secretion from pancreatic β-cells are tightly regulated; their deregulation causes diabetes. Here we map INS-associated loci in human pancreatic islets by 4C and 3C techniques and show that the INS gene physically interacts with the SYT8 gene, located over 300 kb away. This interaction is elevated by glucose and accompanied by increases in SYT8 expression. Inactivation of the INS promoter by promoter-targeting siRNA reduces SYT8 gene expression. SYT8-INS interaction and SYT8 transcription are attenuated by CTCF depletion. Furthermore, SYT8 knockdown decreases insulin secretion in islets. These results reveal a nonredundant role for SYT8 in insulin secretion and indicate that the INS promoter acts from a distance to stimulate SYT8 transcription. This suggests a function for the INS promoter in coordinating insulin transcription and secretion through long-range regulation of SYT8 expression in human islets. View full text Figures at a glance * Figure 1: 4C-Seq analysis reveals the association of SYT8 with INS gene in human pancreatic islets. (–) 4C-Seq analysis of INS-associated loci in the entire human chromosome 11 (), the INS nearby region () and the SYT8-TNNI2 gene locus (). 4C peaks are shown (–), and INS, SYT9, SYT8, SYT13 and SYT7 genes and the H19 ICR are located (,). The frequency of two 4C peaks marked with asterisks is larger than 25,000 and truncated. Panel shows the location of SYT8 and TNNI2 as well as CTSD and LSP1 genes, the 3C PCR primers and the corresponding BglII sites (vertical line) in the SYT8 and TNNI2 locus (lower). The 3C PCR primers are numbered. The exons are marked with solid bars and the transcription direction with arrows. Not all of the BglII sites in this region are shown, and there is only one BglII site within the CTSD gene. () TaqMan quantitative 3C analysis of INS interactions with the SYT8-TNNI2 locus in glucose-treated islets, islets-derived hIPCs and primary human fibroblasts. Plotted are the relative interactions in arbitrary units of the INS gene with eight BglII sit! es (upper, not in scale), which are numbered as in and located in the region shown in . The mean ± s.e.m. is shown (n = 8). The sites for the SYT8 promoter and its 3′ downstream site (or the TNNI2 promoter) are marked with arrows. * Figure 2: Glucose stimulates INS interactions with the SYT8-TNNI2 gene locus and increases SYT8 and TNNI2 gene expression in human islets. () TaqMan quantitative 3C analysis of the relative interaction of the INS gene with the SYT8 promoter and its 3′ downstream site (or the TNNI2 promoter), marked with arrows, in islets from two donors with and without glucose treatment for 30 min. The mean ± s.e.m. is shown (n = 8). () TaqMan quantitative 3C analysis of the interaction of the INS gene with the SYT8 promoter in islets from three donors with and without glucose treatment for 1 h. The SYT8-INS interaction is shown relative to that in islets without glucose treatment. The mean ± s.e.m. is shown (n = 8). () Time-course analysis of the interaction of the INS gene with the SYT8 promoter in islets from one other donor treated with glucose for the indicated times. () Quantitative real-time PCR (qRT-PCR) analysis of SYT8, TNNI2, SYT7, SYT13 and INS gene expression in islets from three donors with and without glucose treatment for the indicated times. The RNA levels are normalized to those of HPRT1, and the mRNA lev! els relative to islets at t = 0 are plotted. The mean ± s.e.m. is shown (n = 9). * Figure 3: The INS promoter positively regulates SYT8 and TNNI2 gene expression in human islets. () qRT-PCR analysis of INS preRNA E2I2 and mature transcript INS E2 (exon 2) and mature transcripts of SYT8, TNNI2, CTSD, MRPL23 and SYT13 genes in islets from two donors that were treated with non-targeting control siRNA (white bar) or one of the two siRNAs targeting to the INS promoter (black bar). The RNA levels are normalized to those of HPRT1. mRNA levels are plotted relative to control. The mean ± s.e.m. is shown (n = 9). The INS exons are shown and the siRNA-targeting sites are marked with arrows (top). () qRT-PCR analysis as in of the islets from one other donor that were treated separately with either of the two siRNAs targeting to the INS promoter. The mean ± s.e.m. is shown (n = 9). * Figure 4: CTCF positively regulates SYT8 and TNNI2 gene expression in human islets and is important for the maintenance of the SYT8-INS interaction in human islets and human fibroblasts. () qRT-PCR analysis of CTCF, SYT8, TNNI2, SYT7 and INS gene expression in islets from four donors that were treated with non-targeting control or CTCF-specific siRNA. The RNA levels are normalized to those of HPRT1. Plotted are the mRNA levels relative to control. () TaqMan quantitative 3C analysis of INS-SYT8 interaction in the two same siRNA-treated islets as shown in . The SYT8-INS interaction is shown relative to control. ***P < 0.0001. () qRT-PCR analysis of CTCF gene expression in normal human primary fibroblasts that were treated with control (white bar) or CTCF-specific siRNA (black bar). Shown are the CTCF mRNA levels relative to control. () Quantitative ChIP analysis of CTCF occupancy at the SYT8 promoter and the INS 3′ downstream region in human fibroblasts treated as in . Shown is the CTCF occupancy relative to control. () TaqMan quantitative 3C analysis of INS-SYT8 interaction in human fibroblasts treated as in . Shown is the SYT8-INS interaction relative to c! ontrol. ***P < 0.0001. The mean ± s.e.m. is shown (n = 8 for and , n = 9 for , and .) * Figure 5: SYT8 is an important regulator of insulin secretion in human islets. () qRT-PCR analysis of SYT8 gene expression in islets treated with control (white bar) or SYT8-specific siRNA (black bar). Total RNA was prepared directly from siRNA-treated islets without medium change. The mean ± s.e.m. is shown (n = 9). () ELISA analysis of insulin levels in the medium for islets from five donors that were separately treated with siRNA as in . The insulin levels are normalized to the amount of total RNAs made from the treated islets. As insulin levels vary among islets from different donors, the insulin levels relative to control are plotted. The mean ± s.e.m. is shown (n = 20, four measures for each of five donors); ***P < 0.0001. () ELISA analysis of insulin levels in the medium of islets from one donor treated as in and cultured with 30.5 mM glucose for 30 min. The insulin levels are normalized to the amount of islets necessary to produce 1 μg total RNA; ***P < 0.0001. () ELISA analysis of insulin levels in the medium of islets from another donor th! at were treated as in and cultured with 30.5 mM glucose or 20 mM L-arginine for 30 min. The insulin levels are normalized as in . ***P < 0.0001; *P = 0.0018. () Insulin secretion from siRNA-treated islets that were treated with 30.5 mM glucose; supernatants were collected every 5 min and tested. The mean ± s.d. is shown for and (n = 4). Author information * Abstract * Author information * Supplementary information Affiliations * Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA. * Zhixiong Xu & * Gary Felsenfeld * Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA. * Gang Wei, * Iouri Chepelev & * Keji Zhao Contributions Z.X. and G.F. designed the experiments; Z.X. conducted the experiments; G.W., I.C. and K.Z. conducted and analyzed Solexa DNA Sequencing experiments; Z.X. and G.F. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Gary Felsenfeld Author Details * Zhixiong Xu Search for this author in: * NPG journals * PubMed * Google Scholar * Gang Wei Search for this author in: * NPG journals * PubMed * Google Scholar * Iouri Chepelev Search for this author in: * NPG journals * PubMed * Google Scholar * Keji Zhao Search for this author in: * NPG journals * PubMed * Google Scholar * Gary Felsenfeld Contact Gary Felsenfeld 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 1–12 and Supplementary Notes Additional data
• MutS switches between two fundamentally distinct clamps during mismatch repair
  - Nat Struct Mol Biol 18(3):379-385 (2011)
  Nature Structural & Molecular Biology | Article MutS switches between two fundamentally distinct clamps during mismatch repair * Cherlhyun Jeong1, 8 * Won-Ki Cho1, 8 * Kyung-Mi Song2 * Christopher Cook3 * Tae-Young Yoon4, 5 * Changill Ban2 * Richard Fishel3, 6 * Jong-Bong Lee1, 7 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:379–385Year published:(2011)DOI:doi:10.1038/nsmb.2009Received16 September 2010Accepted19 November 2010Published online30 January 2011Corrected online13 February 2011 Abstract * Abstract * Accession codes * Change history * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Single-molecule trajectory analysis has suggested DNA repair proteins may carry out a one-dimensional (1D) search on naked DNA encompassing >10,000 nucleotides. Organized cellular DNA (chromatin) presents substantial barriers to such lengthy searches. Using dynamic single-molecule fluorescence resonance energy transfer, we determined that the mismatch repair (MMR) initiation protein MutS forms a transient clamp that scans duplex DNA for mismatched nucleotides by 1D diffusion for 1 s (~700 base pairs) while in continuous rotational contact with the DNA. Mismatch identification provokes ATP binding (3 s) that induces distinctly different MutS sliding clamps with unusual stability on DNA (~600 s), which may be released by adjacent single-stranded DNA (ssDNA). These observations suggest that ATP transforms short-lived MutS lesion scanning clamps into highly stable MMR signaling clamps that are capable of competing with chromatin and recruiting MMR machinery, yet are recycled by ! ssDNA excision tracts. View full text Figures at a glance * Figure 1: Single-molecule FRET of Taq MutS on duplex DNA. () Crystal structure of homodimer Taq MutS bound to unpaired dT showing peptide domains I–V (PDB 1EWQ)42. Donor Cy3 was conjugated to Cys469 of Taq MutS (C42A T469C). () Schematic representation of smFRET assay. Cy5-labeled matched DNA molecules (74 bp) were immobilized on a quartz surface via a biotin-streptavidin linker and the open end blocked anti-digoxigenin (anti-dig). () Representative traces of fluorescence intensity and FRET efficiency for matched DNA molecules in the presence of 10 nM MutS and 100 mM KCl. () Distributions of binding lifetime and dissociation time for 10 nM MutS in 100 mM KCl. A single exponential with mean ± s.e.m. fit the distribution. * Figure 2: Taq MutS scans duplex DNA by rotational diffusion. () FRET efficiency determined from a population histogram of 74-bp duplex DNA molecules at a time resolution of 30 ms. () FRET efficiency determined from a population histogram of 100-bp duplex DNA molecules at a time resolution of 30 ms and 4 ms, respectively. () Average FRET value from individual traces of MutS diffusion on duplex DNA. The Gaussian distribution of FRET efficiency led to the refined FRET efficiency values shown on the 74-bp duplex DNA (n = 78) and on the 100-bp duplex DNA (n = 89) (left). For the transitional diffusion model, the circumference distance between Cy3 and Cy5 determined by the random initial binding remains constant during MutS diffusion. Distributions of FRET efficiency were obtained from 100 trials in silico of the arbitrary binding position of MutS for the 74-bp (red) and 100-bp (blue) duplex DNAs, which evenly range from 0.291 to 0.655 and 0.199 to 0.443, respectively (right). In contrast, the circumference distance varies with rotational d! iffusion in the rotational diffusion model (middle). The resulting distribution of FRET values is a Gaussian with sharp peaks. Illustrations above panels show diffusion models. () The Kd for DNA with blocked ends was determined from the intercept of τduplex•on and τduplex•off for MutS binding to duplex DNA. () The Kd with open-ended DNA was determined from the intercept of τduplex•on and τduplex•off for MutS binding to 3′-unblocked duplex DNA. Illustration is schematic representative of unblocked DNA substrate. * Figure 3: Single-molecule FRET of Taq MutS binding to a +dT mismatch. () Schematic of mismatched DNA molecules containing a single unpaired +dT. Cy5 is positioned at the ninth base from the +dT mismatch. Yellow, nucleotides that contact MutS residues during mismatch binding41, 42, 43. Dig, digoxigenin; anti-dig, antibody to dig. () Representative traces of fluorescence intensity and FRET value for a +dT mismatch in the presence of 10 nM MutS and 100 mM KCl. () FRET efficiency when MutS was bound to the +dT mismatch. () Distributions of MutS binding lifetime and dissociation time for 10 nM MutS in 100 mM KCl. A single exponential with mean ± s.e.m. fit the distribution. () On-rate (k+dT•on = 1 / τ+dT•off) and off-rate (k+dT•off = 1 / τ+dT•on) versus concentration of Taq MutS. The Kd was determined from the intercept of τduplex•on and τduplex•off for MutS binding to a +dT mismatch. () Representative trace of fluorescent intensity and FRET value showing the searching kinetics followed by binding kinetics for a +dT mismatch. () FR! ET efficiency when MutS is searching for a mismatch. () Distributions of binding lifetime for MutS in search of a mismatch at a time resolution of 30 ms. () Distributions of binding lifetime for MutS in search of a mismatch at a time resolution of 4 ms. () Frequency of single molecules where MutS is found searching for a mismatch at a resolution of 30 ms and 4 ms. * Figure 4: ATP induces a long-lived FRET state of MutS. () Schematic representation of ATP or ATPγS effects on mismatch-bound MutS and representative traces of fluorescence intensity and FRET value for a +dT mismatch in the presence of 10 nM MutS, 100 mM KCl and 200 μM ATP. () High FRET efficiency and intermediate FRET efficiency determined from a population histogram of +dT mismatched DNA molecules. () Distributions of MutS binding lifetime to a +dT mismatch in the presence of 1 mM ATP. () Representative time-lapse trace of ATP-bound Taq MutS on the +dT mismatched DNA substrate. () Dwell time of the intermediate FRET state of MutS in the presence of ATP and ATPγS determined from a single exponential of a population histogram of +dT molecules. () The frequency of one, two and three MutS sliding clamps (Cy3) found on 100-bp and 74-bp single DNA molecules in the presence of 30 nM or 300 nM MutS. * Figure 5: Single-stranded DNA provokes the release of ATP-bound MutS sliding clamps. () Representative traces of fluorescence intensity and FRET value for a +dT mismatch containing a (dT)10 ssDNA 5′ tail in the presence of 10 nM MutS, 100 mM KCl and 200 μM ATP. Schematic representation of +dT mismatch containing a (dT)10 ssDNA 5′ tail with MutS and ATP or ATPγS. () Distributions of FRET efficiency and binding lifetime determined from a population histogram of +dT-(dT)10 DNA molecules. () Distributions of FRET efficiency and binding lifetime determined from a population histogram of +dT-(dT)10 DNA molecules in the presence of 200 μM ATP. () Distributions of binding lifetime determined from a histogram of +dT-(dT)10 DNA molecules in the presence of 200 μM ATPγS. * Figure 6: Role of distinct MutS clamps in molecular switch model for MMR. () MutS searching clamps. () MutS mismatch binding and sliding clamps. () MutS–MutL complexes with an MMR excision tract that provokes the release of MutS sliding clamps. Accession codes * Abstract * Accession codes * Change history * Author information * Supplementary information Referenced accessions Protein Data Bank * 1EWQ * 1EWQ Change history * Abstract * Accession codes * Change history * Author information * Supplementary informationCorrigendum 13 February 2011In the version of this article initially published online, the x axis for the right-hand graph in Figure 5c and the x axis for the graph in Figure 5d should have read 'Dwell time (s)'. The error has been corrected in all versions of this article. Author information * Abstract * Accession codes * Change history * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Cherlhyun Jeong & * Won-Ki Cho Affiliations * Department of Physics, Pohang University of Science and Technology (POSTECH), Pohang, Korea. * Cherlhyun Jeong, * Won-Ki Cho & * Jong-Bong Lee * Department of Chemistry, POSTECH, Pohang, Korea. * Kyung-Mi Song & * Changill Ban * Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, Ohio, USA. * Christopher Cook & * Richard Fishel * Department of Physics, Korea Advanced Institute of Science and Technology (KAIST), Daejon, Korea. * Tae-Young Yoon * Institute for the Biocentury, KAIST, Daejon, Korea. * Tae-Young Yoon * Physics Department, The Ohio State University, Columbus, Ohio, USA. * Richard Fishel * School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, Korea. * Jong-Bong Lee Contributions C.J., R.F. and J.-B.L. designed the experiments. C.B. and K.-M.S. contributed essential reagents. C.J. and W.-K.C. carried out single-molecule analysis. C.C. carried out bulk analysis. C.J., W.-K.C., T.-Y.Y., C.B., R.F. and J.-B.L. analyzed the data. C.J., J.-B.L. and R.F. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Jong-Bong Lee or * Changill Ban or * Richard Fishel Author Details * Cherlhyun Jeong Search for this author in: * NPG journals * PubMed * Google Scholar * Won-Ki Cho Search for this author in: * NPG journals * PubMed * Google Scholar * Kyung-Mi Song Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher Cook Search for this author in: * NPG journals * PubMed * Google Scholar * Tae-Young Yoon Search for this author in: * NPG journals * PubMed * Google Scholar * Changill Ban Contact Changill Ban Search for this author in: * NPG journals * PubMed * Google Scholar * Richard Fishel Contact Richard Fishel Search for this author in: * NPG journals * PubMed * Google Scholar * Jong-Bong Lee Contact Jong-Bong Lee Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Change history * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–9 and Supplementary Tables 1 and 2 Additional data
• Substrate-induced remodeling of the active site regulates human HTRA1 activity
  - Nat Struct Mol Biol 18(3):386-388 (2011)
  Nature Structural & Molecular Biology | Brief Communication Substrate-induced remodeling of the active site regulates human HTRA1 activity * Linda Truebestein1 * Annette Tennstaedt1 * Timon Mönig2 * Tobias Krojer3 * Flavia Canellas3 * Markus Kaiser2 * Tim Clausen3 * Michael Ehrmann1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:386–388Year published:(2011)DOI:doi:10.1038/nsmb.2013Received03 May 2010Accepted22 November 2010Published online06 February 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Crystal structures of active and inactive conformations of the human serine protease HTRA1 reveal that substrate binding to the active site is sufficient to stimulate proteolytic activity. HTRA1 attaches to liposomes, digests misfolded proteins into defined fragments and undergoes substrate-mediated oligomer conversion. In contrast to those of other serine proteases, the PDZ domain of HTRA1 is dispensable for activation or lipid attachment, indicative of different underlying mechanistic features. View full text Figures at a glance * Figure 1: Structure of HTRA1. () Structure of HTRA1 trimer. Ribbon presentation of HTRA1 (inactive structure) in top and side views; monomers are shown in different colors. () Structures of active and inactive HTRA1. For clarity, the inhibitor bound to the active protease is not shown. The active-site residues are shown in stick mode, and functional loops are highlighted. Bottom, stereo presentation of the superimposed active and inactive HTRA1 conformations. () Catalytic triads. Aligned catalytic triads of the active (yellow) and inactive (gray) HTRA1. The inactive conformation shows a distorted catalytic triad, with residue Ser328 (here replaced by alanine) being too distant from His220 (9.5Å) for proton transfer. * Figure 2: HTRA1 in complex with peptide inhibitor. () HtrA1 trimer with inhibitor. Ribbon presentation of the HTRA1 trimer in bottom view with bound inhibitor (blue). () Electron density map of inhibitor. Stereo view of the Fo − Fc electron density map of the DPMFKLboroV inhibitor that was calculated at 2.8-Å resolution without ligand and contoured at 3σ. The active-site residues (green) and the inhibitor molecule (P1–P5) (blue) are shown in stick presentation. Residues in loop L3 forming hydrophobic interactions with the inhibitor molecule are shown in stick presentation (orange). () Interactions of inhibitor with HTRA1. Schematic view of interactions between protein and inhibitor. (1) P4-Phe and P2-Leu form hydrophobic interactions with HTRA1; (2), (3) and (4) indicate hydrogen bonds to HTRA1 backbone; (5) indicates binding of the boronate to the active-site Ser328; (6) P1-Val points into S1 specificity pocket. P6-Pro and P7-Asp have been omitted from the model, and the side chain of P4-Lys could not be modeled. * Figure 3: Role of the PDZ domain and activation by oligomerization. () Casein digests. Activity of HTRA1 and HTRA1prot with casein as substrate. Samples were taken at the time points indicated and subjected to SDS-PAGE. () Activation of HTRA1 by denatured citrate synthase (CS). Specific activity of HTRA1 or HTRA1prot was determined using VFNTLPMMGKASPV-pNA as a substrate with or without denatured CS by measuring the change of absorbance at 405 nm (AU, absorbance units). () Change of oligomeric state. HTRA1 (green), CS (blue) and HTRA1 incubated with heat-denatured CS (black) were fractionated by size exclusion chromatography. Peak fractions were analyzed by SDS-PAGE. Accession codes * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3NUM * 3NWU * 3NZI * 3NUM * 3NWU * 3NZI Author information * Accession codes * Author information * Supplementary information Affiliations * Centre for Medical Biotechnology, Faculty of Biology, University Duisburg–Essen, Essen, Germany. * Linda Truebestein, * Annette Tennstaedt & * Michael Ehrmann * Chemical Genomics Centre, Max-Planck-Gesellschaft, Dortmund, Germany. * Timon Mönig & * Markus Kaiser * Research Institute for Molecular Pathology (IMP), Vienna, Austria. * Tobias Krojer, * Flavia Canellas & * Tim Clausen Contributions L.T., A.T., T.M. and F.C. performed experiments; L.T. and T.K. solved the structures; L.T., M.K., T.C. and M.E. planned the experiments and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Michael Ehrmann or * Tim Clausen Author Details * Linda Truebestein Search for this author in: * NPG journals * PubMed * Google Scholar * Annette Tennstaedt Search for this author in: * NPG journals * PubMed * Google Scholar * Timon Mönig Search for this author in: * NPG journals * PubMed * Google Scholar * Tobias Krojer Search for this author in: * NPG journals * PubMed * Google Scholar * Flavia Canellas Search for this author in: * NPG journals * PubMed * Google Scholar * Markus Kaiser Search for this author in: * NPG journals * PubMed * Google Scholar * Tim Clausen Contact Tim Clausen Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Ehrmann Contact Michael Ehrmann Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–4, Supplementary Notes, Supplementary Table 1 and Supplementary Methods Additional data
• Structural basis of signal-sequence recognition by the signal recognition particle
  - Nat Struct Mol Biol 18(3):389-391 (2011)
  Nature Structural & Molecular Biology | Brief Communication Structural basis of signal-sequence recognition by the signal recognition particle * Tobias Hainzl1 * Shenghua Huang1 * Gitte Meriläinen1 * Kristoffer Brännström2 * A Elisabeth Sauer-Eriksson1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:389–391Year published:(2011)DOI:doi:10.1038/nsmb.1994Received25 August 2010Accepted01 December 2010Published online20 February 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The signal recognition particle (SRP) recognizes and binds the signal sequence of nascent proteins as they emerge from the ribosome. We present here the 3.0-Å structure of a signal sequence bound to the Methanococcus jannaschii SRP core. Structural comparison with the free SRP core shows that signal-sequence binding induces formation of the GM-linker helix and a 180° flip of the NG domain—structural changes that ensure a hierarchical succession of events during protein targeting. View full text Figures at a glance * Figure 1: Structure of the M. jannaschii S domain of SRP in complex with a functional signal sequence. () The fused signal sequence promotes SRP–SRP receptor interaction. GTPase rates of SRP–SRP receptor complexes were determined in multiple turnover reactions in the presence of 0.5 μM M. jannaschii SRP S domains and varying concentrations of M. jannaschii SR93–408. Curves are shown for the S domain containing full-length SRP54 (blue), C-terminally truncated SRP541–427 (brown), SRP54–ss (green) and an aspartate mutant (SRP54–ss (L446D L450D); red). In addition, the curve for SRP54–ss without SRP RNA (black) is shown. () Secondary structure and nucleotide sequence of the S-domain SRP RNA used for crystallization are shown, with the numbering corresponding to full-length M. jannaschii SRP RNA. Lines between bases indicate Watson-Crick base pairs; black circles, noncanonical pairs; and red circles, tertiary RNA–RNA interactions. Highlighted in pink are the conserved loop motifs: tetraloop, symmetric and asymmetric loops, and loop E. The approximate positions of ! the binding sites for SRP54 (M domain, green; N domain, cyan; and G domain, dark blue) and SRP19 (orange) are indicated. The signal sequence (ss) is indicated in yellow and the GM linker in magenta. () Ribbon representations of the SRP–signal sequence complex with the color code as in . * Figure 2: Structural basis for signal-sequence recognition. () Detailed view of the signal sequence in the binding pocket. The first two turns of the signal sequence pack against the GM-linker helix and α-M1 to form a hydrophobic core. () Surface representation of the signal-sequence binding site, colored according to electrostatic potential (blue is positive, red is negative and white is neutral). The signal sequence is shown as stick-and-ball. * Figure 3: Signal-sequence induced structural changes in the SRP core. The M. jannaschii S domain in the free (left) (2V3C15) and signal sequence–bound (right) forms of SRP. Formation of the signal sequence–bound form depends on a 90° rotation and a 180° flip of the NG domain, which brings the G domain into proximity of the helix 8 tetraloop. Accession codes * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 3NDB * 3NDB Author information * Accession codes * Author information * Supplementary information Affiliations * Department of Chemistry, Umeå University, Umeå, Sweden. * Tobias Hainzl, * Shenghua Huang, * Gitte Meriläinen & * A Elisabeth Sauer-Eriksson * Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden. * Kristoffer Brännström Contributions T.H., S.H. and A.E.S.-E. contributed to every aspect of this work. G.M. contributed to cloning and protein production and K.B. to Biacore experiments. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Tobias Hainzl Author Details * Tobias Hainzl Contact Tobias Hainzl Search for this author in: * NPG journals * PubMed * Google Scholar * Shenghua Huang Search for this author in: * NPG journals * PubMed * Google Scholar * Gitte Meriläinen Search for this author in: * NPG journals * PubMed * Google Scholar * Kristoffer Brännström Search for this author in: * NPG journals * PubMed * Google Scholar * A Elisabeth Sauer-Eriksson Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (796K) Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Methods Additional data
• Retinal dynamics underlie its switch from inverse agonist to agonist during rhodopsin activation
  - Nat Struct Mol Biol 18(3):392-394 (2011)
  Nature Structural & Molecular Biology | Brief Communication Retinal dynamics underlie its switch from inverse agonist to agonist during rhodopsin activation * Andrey V Struts1, 2 * Gilmar F J Salgado3 * Karina Martínez-Mayorga4 * Michael F Brown1, 5 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:392–394Year published:(2011)DOI:doi:10.1038/nsmb.1982Received09 June 2010Accepted16 November 2010Published online30 January 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg X-ray and magnetic resonance approaches, though central to studies of G protein–coupled receptor (GPCR)-mediated signaling, cannot address GPCR protein dynamics or plasticity. Here we show that solid-state 2H NMR relaxation elucidates picosecond-to-nanosecond–timescale motions of the retinal ligand that influence larger-scale functional dynamics of rhodopsin in membranes. We propose a multiscale activation mechanism whereby retinal initiates collective helix fluctuations in the meta I–meta II equilibrium on the microsecond-to-millisecond timescale. View full text Figures at a glance * Figure 1: Site-specific 2H NMR relaxation illuminates functional dynamics of retinylidene methyl groups within binding pocket of rhodopsin. () Light absorption yields cis-to-trans isomerization at position 11, converting retinal from an inverse agonist to an agonist through series of rhodopsin intermediates (designated photo, batho, blue‐ shifted intermediate (BSI), lumi, meta I, meta II) with different time scales. (–) Solid-state 2H NMR spectra for dark-state rhodopsin with 11-cis-retinal deuterated at C5, C9 or C13 with C2H3 groups in POPC bilayers (1:50 molar ratio). The 2H NMR line shapes indicate rapid axial spinning of C−C2H3 groups down to at least −160 °C. (,) Partially relaxed 2H NMR spectra for retinylidene C9- and C13-Me groups of rhodopsin in aligned POPC membranes (θ = 0°) at −150 °C. () Inversion-recovery plots showing site-specific variations in spin-lattice (T1Z) relaxation times for C9- and C13-Me groups at −150 °C. * Figure 2: Solid-state 2H NMR captures site-specific changes in retinal mobility during light activation of rhodopsin. (–) Spin-lattice (T1Z) relaxation times (±s.d.) of retinylidene methyl groups are shown versus reciprocal temperature in the dark (), meta I () and meta II () states (−30 to −160 °C). Methyl dynamics are described by an axial three-fold jump model or a continuous diffusion model with coefficients D|| and D⊥. In –, rotation about the methyl threefold (C3) axis corresponds to solid lines with D⊥ = 0; the dashed lines include restricted off-axial diffusion (D⊥ = D||). Fits for the C5-Me in meta I in assume unlike rotational diffusion constants (D|| ≠ D⊥) (dashed line) or the presence of two conformers with different bond orientations and axial diffusion coefficients (solid line). * Figure 3: 2H NMR relaxation of retinal sheds new light on activation mechanism of rhodopsin. () Summary of analysis of solid-state 2H NMR measurements. Order parameters of rapidly spinning methyl groups are designated by SC3; the pre-exponential factor is k0 for three-fold axial jumps or D0 for continuous diffusion; and Ea indicates the activation energy. (The diffusion model assumes either D⊥ = 0 (right) or ηD ≡ D||/D⊥ = 1 (left) except for the C5-Me in meta I, where ηD ≠ 1.) (–) Proposed activation mechanism for rhodopsin in membranes based on X-ray19, FTIR11 and 2H NMR data12. Isomerization of retinal displaces the E2 loop toward the extracellular (e) side, with fluctuations of helices H5 and H6 exposing transducin (Gt) recognition sites on the opposing cytoplasmic (c) surface. Figure produced (PDB 1U19)3 using PyMOL (http://pymol.sourceforge.net/). Accession codes * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 1U19 * 1U19 Author information * Accession codes * Author information * Supplementary information Affiliations * Department of Chemistry, University of Arizona, Tucson, Arizona, USA. * Andrey V Struts & * Michael F Brown * Department of Physics, St. Petersburg State University, St. Petersburg, Russia. * Andrey V Struts * Département de Chimie, École Normale Supérieure, Paris, France. * Gilmar F J Salgado * Torrey Pines Institute for Molecular Studies, Fort Pierce, Florida, USA. * Karina Martínez-Mayorga * Department of Physics, University of Arizona, Tucson, Arizona, USA. * Michael F Brown Contributions A.V.S. and M.F.B. designed the research. A.V.S. and G.F.J.S. performed the experiments. A.V.S., G.F.J.S., and K.M.-M. analyzed the data. A.V.S. and M.F.B. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael F Brown Author Details * Andrey V Struts Search for this author in: * NPG journals * PubMed * Google Scholar * Gilmar F J Salgado Search for this author in: * NPG journals * PubMed * Google Scholar * Karina Martínez-Mayorga Search for this author in: * NPG journals * PubMed * Google Scholar * Michael F Brown Contact Michael F Brown Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Methods and Supplementary Figures 1 and 2 Additional data
• Genome-wide mapping of Arabidopsis thaliana origins of DNA replication and their associated epigenetic marks
  - Nat Struct Mol Biol 18(3):395-400 (2011)
  Nature Structural & Molecular Biology | Resource Genome-wide mapping of Arabidopsis thaliana origins of DNA replication and their associated epigenetic marks * Celina Costas1, 7 * Maria de la Paz Sanchez1, 6, 7 * Hume Stroud2, 7 * Yanchun Yu3 * Juan Carlos Oliveros4 * Suhua Feng5 * Alberto Benguria4 * Irene López-Vidriero4 * Xiaoyu Zhang3 * Roberto Solano4 * Steven E Jacobsen2, 5 * Crisanto Gutierrez1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:395–400Year published:(2011)DOI:doi:10.1038/nsmb.1988Received19 February 2010Accepted24 November 2010Published online06 February 2011 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 Genome integrity requires faithful chromosome duplication. Origins of replication, the genomic sites at which DNA replication initiates, are scattered throughout the genome. Their mapping at a genomic scale in multicellular organisms has been challenging. In this study we profiled origins in Arabidopsis thaliana by high-throughput sequencing of newly synthesized DNA and identified ~1,500 putative origins genome-wide. This was supported by chromatin immunoprecipitation and microarray (ChIP-chip) experiments to identify ORC1- and CDC6-binding sites. We validated origin activity independently by measuring the abundance of nascent DNA strands. The midpoints of most A. thaliana origin regions are preferentially located within the 5′ half of genes, enriched in G+C, histone H2A.Z, H3K4me2, H3K4me3 and H4K5ac, and depleted in H3K4me1 and H3K9me2. Our data help clarify the epigenetic specification of DNA replication origins in A. thaliana and have implications for other eukaryotes. View full text Figures at a glance * Figure 1: Identification of DNA replication origins in the A. thaliana genome. () Representative genome-browser view of a region in chromosome 1. Genes (green) transcribed from each strand are along the chromosome above and below the position scale. Bottom, an enlarged region containing a replication origin, determined as a region enriched for BrdU-labeled DNA strands (light blue) relative to the unlabeled control DNA (black), together with the ORC1 (red) and CDC6 (dark blue) binding patterns (posterior probabilities for ORC1 and CDC6 data sets). Origins, for example, ori1-0850, are named based on their chromosomal location (ori1 through ori5), followed by the four digits indicating the origin number within each chromosome. They are named consecutively starting at the left tip of each chromosome; that is, for chromosome 1, in which we identified 376 origins, the leftmost origin is ori1-0010 and the rightmost one is ori1-3760. () The pattern of ORC1 and CDC6 binding over origin regions was obtained by plotting their relative binding signal ±10 kb from ! the origin region midpoint (0) using 50-bp sliding windows (smoothed). The P values (two-sided) of the difference in the ChIP-chip signals in origins (midpoint ±300 bp), using a two-tailed Welch test, were 7.25 × 10−6 and 1.29 × 10−10 for ORC1 and CDC6, respectively. () Number of origins relative to chromosomal size. Chromosome size (relative to chromosome (chr.) 1) versus the number of origins identified in each chromosome (relative to origin number in chromosome 1). Number of origins identified in each chromosome is in parentheses. () Distribution of interorigin distances, measured as distance between the midpoints of two contiguous origins (median, 51.1 kb; average, 77.2 kb; s.d., 83.4 kb). * Figure 2: DNA replication origin activity determined by nascent DNA strand abundance. () Several putative origin-containing regions were chosen for detailed measurement by real-time PCR of nascent strand abundance in a sample of short DNA molecules containing an RNA primer at their 5′ end (see Online Methods). Genomic region under study is at bottom of each panel and shows the location of genes (green), ORC1-binding (red) and CDC6-binding (dark blue) signals, and putative origin location (light blue), defined by direct sequencing of the BrdU-labeled DNA sample (see Online Methods). DNA fragments (~200 bp long) amplified by primer pairs scanning each region are small black rectangles on the x axis. Coordinates in each chromosome are at the bottom of each panel. Results correspond to PCR amplifications using fraction 5 (see Online Methods). Data for origins ori1-2300 (), ori2-1340 () and ori2-1430 (). Data for a region used as a negative control around gene at4g14700 that lacks BrdU-labeled DNA sequences (). * Figure 3: Genomic location of A. thaliana replication origins. () Percentage of origins colocalizing with various genomic elements. Numbers in parentheses, proportion of A. thaliana genome represented by each class. () Origin densities were computed for regions upstream, downstream and within genes of different expression levels (all genes, highest 25%, lowest 25%). Regions 2 kb upstream and downstream of genes, as well as the bodies of genes, were each divided into ten bins, and the origin densities (origins per 106 bp) were calculated for each bin and represented as box plots. White lines, median; edges of boxes, 25th (bottom) and 75th (top) percentiles; error bars, minimum and maximum points that fell within 1.5×IQR (interquartile range) below the 25th percentile or above the 75th percentile. * Figure 4: Relationship of A. thaliana replication origins to CG methylation and histone H2A.Z. () Relative levels of CG, CHG and CHH methylation ±10 kb relative to the origin midpoint (0) in 50-bp sliding windows (smoothed). Methylation data have been reported19. () G+C content (%) of replication origins (black) and indicated genomic regions (white). These values were calculated from the sequence data files available at The Arabidopsis Information Resource (TAIR), http://www.arabidopsis.org/. () Density of the histone variant H2A.Z in a ±10 kb region relative to the origin midpoint (0) in 50-bp sliding windows (smoothed). The genomic distribution of H2A.Z has been reported32. The P value of the difference in the ChIP-chip signals in origins (calculated as in Fig. 1b; see Online Methods), was 9.34 × 10−34. * Figure 5: Histone modification landscape around replication origins. () Relative level of indicated histone mark ±10 kb relative to the center of origins (0) in 50-bp sliding windows (smoothed). Data for H3K4me and H3K9me2 have been reported20, 21. The P values of the difference in the ChIP-chip signals in origins (calculated as in Fig. 1b; see Online Methods) were 0.86, 3.52 × 10−28, 1.07 × 10−41 and 7.33 × 10−14 for H3K4me1, H3K4me2, H3K4me3 and H3K9me2, respectively. () Relationship between H3K4 methylation status and the presence of origins. We calculated the fraction of genes containing origins and different combinations of H3K4 methylation, and compared it with the fraction of all genes containing the same H3K4me combinations21. Different classes are ordered with decreasing values of the fraction of genes with origins. () Relative level of H4K5ac ±10 kb relative to the center of origins (0) in 50-bp sliding windows (smoothed). Calculations are based on the ChIP-chip data set generated in this work. The P value of the differen! ce in the ChIP-chip signals in origins (calculated as in 1b; see Online Methods) was 1.23 × 10−23. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE21928 * GSE21828 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Celina Costas, * Maria de la Paz Sanchez & * Hume Stroud Affiliations * Centro de Biologia Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad autónoma de Madrid (UAM), Madrid, Spain. * Celina Costas, * Maria de la Paz Sanchez & * Crisanto Gutierrez * Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, California, USA. * Hume Stroud & * Steven E Jacobsen * Department of Plant Biology, University of Georgia, Athens, Georgia, USA. * Yanchun Yu & * Xiaoyu Zhang * Centro Nacional de Biotecnología, CSIC, Madrid, Spain. * Juan Carlos Oliveros, * Alberto Benguria, * Irene López-Vidriero & * Roberto Solano * Howard Hughes Medical Institute, University of California, Los Angeles, California, USA. * Suhua Feng & * Steven E Jacobsen * Present address: Instituto de Ecología, Universidad Nacional Autónoma de México, Mexico DF, Mexico. * Maria de la Paz Sanchez Contributions C.C., M.P.S., Y.Y., S.F., A.B. and I.L.-V. carried out experiments. H.S., J.C.O., C.C., M.P.S., X.Z. and R.S. analyzed data. C.G. and S.E.J. prepared the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Crisanto Gutierrez or * Steven E Jacobsen Author Details * Celina Costas Search for this author in: * NPG journals * PubMed * Google Scholar * Maria de la Paz Sanchez Search for this author in: * NPG journals * PubMed * Google Scholar * Hume Stroud Search for this author in: * NPG journals * PubMed * Google Scholar * Yanchun Yu Search for this author in: * NPG journals * PubMed * Google Scholar * Juan Carlos Oliveros Search for this author in: * NPG journals * PubMed * Google Scholar * Suhua Feng Search for this author in: * NPG journals * PubMed * Google Scholar * Alberto Benguria Search for this author in: * NPG journals * PubMed * Google Scholar * Irene López-Vidriero Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaoyu Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Roberto Solano Search for this author in: * NPG journals * PubMed * Google Scholar * Steven E Jacobsen Contact Steven E Jacobsen Search for this author in: * NPG journals * PubMed * Google Scholar * Crisanto Gutierrez Contact Crisanto Gutierrez Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information Text files * Supplementary Table 1 (432K) Putative Arabidopsis DNA replication origins (originome) PDF files * Supplementary Text and Figures (756K) Supplementary Figures 1–7 and Supplementary Table 2 Additional data