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• In self-defense
  - Nat Struct Mol Biol 18(4):401-402 (2011)
  Nature Structural & Molecular Biology | News and Views In self-defense * Piet Gros1Journal name:Nature Structural & Molecular BiologyVolume: 18,Pages:401–402Year published:(2011)DOI:doi:10.1038/nsmb.2036Published online20 February 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. Discrimination between self and non-self surfaces by the complement system of innate immunity has long been enigmatic. Finally, two papers provide structural insights into host protection against indiscriminate immune surveillance. View full text Figures at a glance * Figure 1: Schematic overview of the amplification of the complement response and host protection by factor H. The left side shows the amplification by the alternative pathway. C3 is cleaved into anaphylatoxin C3a and opsonin C3b. Pro-protease factor (FB) binds to C3b and is then cleaved by factor D (FD), releasing the pro-peptide segment Ba and yielding the C3 convertase complex C3bBb. The right side shows host protection by factor H. Factor H domains 19–20 are highlighted in red and domains 1–4 in orange. Factor H dissociates the convertase into C3b and Bb and is cofactor to factor I (FI) in degrading C3b into iC3b and finally C3d. * Figure 2: Comparison of the C3d–FH19–20 complexes. The structure of the 2:1 complex presented by Kajander et al.3 is shown on the left, and a superposition of the complexes (three 1:1 complexes in the asymmetric unit) as reported by Morgan et al.2 is shown on the right. All complexes show a C3d molecule bound to domain 19 of factor H. Kajander et al. use a double mutation of FH19–20 (D1119G and Q1139A) that strongly reduces the affinity of C3d for the domain 19 site. Nevertheless, the structure of Kajander et al. shows a similar association of C3d to domain 19 as the 3 complexes of Morgan et al., in which the wild-type sequence for FH19–20 is used (the side chains of the two residues Asp1119 and Gln1139 are shown as red spheres). Although all three FH19–20 molecules have one C3d bound to domain 19 in the structure reported by Morgan et al., further analysis of the contacts reveals that in one out of three, FH19–20 has an additional contact to C3d through domain 20, similar to the one observed by Kajander et al. (this! contact is centered on Arg1203). The residues implied in binding glycosaminoglycans, as indicated by the two papers, are shown as gray spheres (on the Cα positions). 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 * Piet Gros is in the Laboratory of Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Utrecht University, Utrecht, The Netherlands. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Piet Gros Author Details * Piet Gros Contact Piet Gros 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(4):403 (2011)
  Nature Structural & Molecular Biology | Research Highlights Research highlights Journal name:Nature Structural & Molecular BiologyVolume: 18,Page:403Year published:(2011)DOI:doi:10.1038/nsmb0411-403Published online06 April 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. Seeing single spliceosomes The spliceosome is a complex macromolecular machine made up of 5 small nuclear RNAs (snRNAs) and ~100 core proteins that act on precursor messenger RNAs (pre-mRNAs) to remove introns and join exons together. Previous work has shown that individual spliceosome subcomplexes associate with pre-mRNA in an ordered and sequential way to form functional spliceosomes. To directly examine the kinetics of subcomplex association with pre-mRNAs, Moore, Gelles, Cornish and colleagues labeled individual subcomplexes with different fluorophores and analyzed their assembly by using a previously published multiwavelength fluorescence technique—colocalization single-molecule spectroscopy (CoSMoS). Experiments were carried out in yeast whole-cell extracts, and the single fluorophore labeled pre-mRNA was tethered to a glass surface. In this system, the appearance and disappearance of fluorescent spots that colocalize with the surface represent the comings and goings of individual spliceosome ! subcomplexes. The authors found that commitment of an individual pre-mRNA to splicing increases as the assembly process proceeds and that each step seems to be reversible. Future studies using single-molecule systems such as this could lead to new insights into the mechanism of pre-mRNA splicing and perhaps alternative splicing as well. (Science331, 1289–1295, 2011) BK ABA remastered Some plant biologists feel there is a bias against their field because their findings are often not deemed directly relevant to human and animal models. Such perceived limitation can be turned to one's advantage, as recently described by Crabtree and colleagues, who co-opted molecular events in a plant signaling pathway to control the proximity of proteins in mammalian cells. Abscisic acid (ABA) is a plant hormone that controls seed dormancy and responses to environmental stress. Resolution of the ABA pathway has seen recent breakthroughs, with the identification of ABA receptors and the development of a mechanistic understanding of the molecular events involved. ABA binds to members of the PYL-PYR-RCAR family of proteins, triggering a conformational change that results in the recruitment and inhibition of phosphatases of the PP2C family such as ABI. This is essentially a dimerization event controlled by ABA. Fu-Sen Liang working in the Crabtree lab fused fragments of the AB! A receptor PYL1 and the phosphatase ABI containing the complementary surfaces to different target proteins that would be in proximity only in the presence of ABA. Similar systems already exist; for example, rapamycin (Rap) can induce the proximity of proteins containing a domain from FKBP and Frb. However, because the ABA pathway does not exist in mammalian cells, there are no endogenous binding proteins that would compete for its availability. Also, unlike rapamycin, ABA has no detectable toxicity and in fact is readily found in fruits and vegetables that we consume. The authors directly compared the Rap and ABA systems in controlling gene activation, and they found a faster and more linear response with ABA. Moreover, ABA had the added benefit of being easier to wash off from cells than Rap. Both Rap and ABA systems can be used in the same cell to control different biological processes increasing the potential applications of both techniques. The authors also assessed the! ABA system for controlling protein localization (with one of ! the fusion partners targeted to the different cellular compartments) and signal transduction events (by directing the membrane localization of Son of Sevenless and hence the activation of GTPase Ras). A catalytically inactive ABI mutant also worked well in this system, which should circumvent any potential issues due to extraneous phosphatase activity in the cell. Finally, ABA is quite inexpensive and was found to be stable in cultured mammalian cells and in mouse serum, even after oral administration; thus, in addition to its value as an experimental tool in cells, the ABA system has the potential to be used in animal models and perhaps even therapeutically. (Sci. Signal.4, rs2, 2011) IC 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
• Structure and VP16 binding of the Mediator Med25 activator interaction domain
  - Nat Struct Mol Biol 18(4):404-409 (2011)
  Nature Structural & Molecular Biology | Article Structure and VP16 binding of the Mediator Med25 activator interaction domain * Erika Vojnic1 * André Mourão2, 3, 4 * Martin Seizl1 * Bernd Simon4 * Larissa Wenzeck1 * Laurent Larivière1 * Sonja Baumli1, 6 * Karen Baumgart5 * Michael Meisterernst5 * Michael Sattler2, 3 * Patrick Cramer1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:404–409Year published:(2011)DOI:doi:10.1038/nsmb.1997Received17 May 2010Accepted03 December 2010Published online06 March 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 Eukaryotic transcription is regulated by interactions between gene-specific activators and the coactivator complex Mediator. Here we report the NMR structure of the Mediator subunit Med25 (also called Arc92) activator interaction domain (ACID) and analyze the structural and functional interaction of ACID with the archetypical acidic transcription activator VP16. Unlike other known activator targets, ACID forms a seven-stranded β-barrel framed by three helices. The VP16 subdomains H1 and H2 bind to opposite faces of ACID and cooperate during promoter-dependent activated transcription in a in vitro system. The activator-binding ACID faces are functionally required and conserved among higher eukaryotes. Comparison with published activator structures reveals that the VP16 activation domain uses distinct interaction modes to adapt to unrelated target surfaces and folds that evolved for activator binding. View full text Figures at a glance * Figure 1: Solution structure of Med25 ACID. () Med25 domain architecture. () Stereo view of the backbone atoms (N, Cα and C′) of ten superimposed lowest-energy NMR structures. () Ribbon model for the lowest-energy conformer of ACID (left) and comparison with the SPOC domain of SHARP42 (PDB 1OW1, right). Helices unique to SPOC are in green. Figures prepared with MOLMOL53 and PyMOL (http://www.pymol.org/). * Figure 2: VP16–ACID interaction. () Sequence of VP16 TAD with subdomains H1 and H2. Regions known to form a helical structure upon target interaction are underlined. Sites of mutation are highlighted with boxes (compare with Fig. 4c). () Overlay of the 2D 1H,15N HSQC spectra for 15N-labeled ACID in free form (black) and in the presence of 1.3 molar excess of VP16 TAD (red). Insets, chemical shift perturbations of specific residues upon addition of 0.2 (blue), 0.6 (purple), 1 (gold) and 1.3 (red) molar equivalents of TAD. () Histogram of the variation in chemical shift (Δδ (p.p.m.)) observed in the 2D 1H,15N HSQC spectrum of ACID upon formation of the ACID–VP16 TAD complex at a molar ratio of 1:1.3. Red lines, residues with signals that were exchange-broadened, corresponding to red spheres in Figure 3 and Supplementary Figure 1. () Mapping of residues that undergo chemical shift changes (Δδ > 0.6 p.p.m.) in the 1H,15N HSQC spectra upon binding of VP16 TAD to the ACID structure. Rising red color intensi! ties correspond to increasing chemical shift changes. () Overlay of the 2D 1H,15N HSQC spectra for 15N-labeled ACID in free form (black) and in the presence of an eight-fold molar excess of VP16 subdomain H2 (red). Insets, chemical shift perturbations of specific residues upon addition of 0.5 (blue), 1.5 (purple), 2 (gold) and 8 (red) molar equivalents of H2. () Histogram as in but for VP16 H2 titration at a ratio of ACID:VP16 H2 of 1:1.5. () Mapping of residues that undergo chemical shift changes (Δδ > 0.6 p.p.m.) as in but for VP16 H2 binding onto the ACID structure. * Figure 3: VP16-binding interface of ACID. Two views of the ACID structure related by a 180° rotation around a vertical axis are shown with residues perturbed upon VP16 binding (top) or as an electrostatic surface representation (bottom, blue and red for positive and negative charges, respectively). Rising red color intensities correspond to increasing chemical shift changes upon binding of VP16-TAD (Fig. 2b–d). Spheres indicate residues with signals that show binding in intermediate exchange (Fig. 2c and Supplementary Fig. 1). Dashed circles indicate opposite ACID faces interacting mainly with H1 and H2, respectively. * Figure 4: Functional ACID-VP16 interaction. () EMSA supershift assay. The complex formed by DNA and Gal4-VP16 (lane 2) underwent a supershift with increasing concentrations of wild-type ACID (lanes 4–6). This supershift was abolished by ACID point mutation R466E (lane 7–9). () ACID quenched VP16 activation in a yeast transcription system. Assays were performed with yeast nuclear extracts (lane 1) or extracts with increasing amounts (10, 100 or 400 pmol) of wild-type ACID (lanes 2–4) or ACID variant R466E (lanes 5–7). Transcription was quenched by recombinant ACID, but not by ACID variant R466E. () VP16 subdomains H1 and H2 activated yeast transcription in a synergistic, rather than competitive, manner. Transcription in yeast nuclear extracts was monitored in the absence (lanes 1, 10) or the presence of different Gal4-VP16 variants, including Gal4 fusions with VP16 TAD (lanes 2, 3, 11, 12), TAD carrying the H1 subdomain mutation F442P (H1mt; lanes 4, 5), H1 (lanes 6, 7), H1mt (lanes 8, 9), TAD carrying the H2 s! ubdomain mutations F473A F475A F479A (H2mt; lanes 13, 14), H2 (lanes 15, 16) and H2mt (lanes 17, 18). () ACID inhibited transcription activation by VP16 in mammalian B-cell nuclear extracts. Assays were performed with nuclear extracts (lane 1) or extracts with increasing amounts (530 or 850 pmol) of wild-type ACID (lanes 2, 3) or specific ACID point mutants (lanes 4–13). The R466E variant of ACID hardly quenched transcription (lanes 4, 5), whereas other ACID variants did to various extents (lanes 6–13). Data are presented as average values of three experiments ± s.d. and one representative gel is shown. * Figure 5: Model of an activated transcription initiation complex. A Pol II initiation complex was modeled on promoter DNA based on published results54, 55, and DNA was extended with B-DNA. Blue, Mediator; orange, activators. * Figure 6: Comparison of ACID with known activator–target complexes. Activators are in orange and target domains are in silver. Depicted are ACID (this study), Tfb1–VP16 (ref. 21), NcoA-1–STAT6 (ref. 46), MDM2–p53 (ref. 47) and CBP-KIX–CREB-pKID (ref. 45). PDB codes are in parentheses underneath each structure. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 2XNF * 2XNF Referenced accessions Protein Data Bank * 1OW1 * 1OW1 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Gene Center and Department of Biochemistry, Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany. * Erika Vojnic, * Martin Seizl, * Larissa Wenzeck, * Laurent Larivière, * Sonja Baumli & * Patrick Cramer * Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany. * André Mourão & * Michael Sattler * Biomolecular NMR and Center for Integrated Protein Science Munich (CIPSM), Department Chemie, Garching, Germany. * André Mourão & * Michael Sattler * European Molecular Biology Laboratory (EMBL), Heidelberg, Germany. * André Mourão & * Bernd Simon * Institute of Molecular Tumor Biology, University of Muenster, Muenster, Germany. * Karen Baumgart & * Michael Meisterernst * Present address: Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, Oxford, UK. * Sonja Baumli Contributions E.V., A.M. and B.S., NMR data acquisition and analysis; E.V., M. Seizl, L.W., L.L. and S.B., sample preparation and functional assays; K.B. and M.M., mammalian transcription assays; M.M., M. Sattler and P.C., project design and supervision; E.V., M. Sattler and P.C., manuscript preparation. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Michael Sattler or * Patrick Cramer Author Details * Erika Vojnic Search for this author in: * NPG journals * PubMed * Google Scholar * André Mourão Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Seizl Search for this author in: * NPG journals * PubMed * Google Scholar * Bernd Simon Search for this author in: * NPG journals * PubMed * Google Scholar * Larissa Wenzeck Search for this author in: * NPG journals * PubMed * Google Scholar * Laurent Larivière Search for this author in: * NPG journals * PubMed * Google Scholar * Sonja Baumli Search for this author in: * NPG journals * PubMed * Google Scholar * Karen Baumgart Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Meisterernst Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Sattler Contact Michael Sattler Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick Cramer Contact Patrick Cramer 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 (545K) Supplementary Figures 1–6 and Supplementary Methods Additional data
• Structure of the VP16 transactivator target in the Mediator
  - Nat Struct Mol Biol 18(4):410-415 (2011)
  Nature Structural & Molecular Biology | Article Structure of the VP16 transactivator target in the Mediator * Alexander G Milbradt1 * Madhura Kulkarni2, 3, 5 * Tingfang Yi1, 5 * Koh Takeuchi1, 4 * Zhen-Yu J Sun1 * Rafael E Luna1 * Philipp Selenko1, 4 * Anders M Näär2, 3 * Gerhard Wagner1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:410–415Year published:(2011)DOI:doi:10.1038/nsmb.1999Received18 May 2010Accepted03 December 2010Published online06 March 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 human Mediator coactivator complex interacts with many transcriptional activators and facilitates recruitment of RNA polymerase II to promote target gene transcription. The MED25 subunit is a critical target of the potent herpes simplex 1 viral transcriptional activator VP16. Here we determine the solution structure of the MED25VP16-binding domain (VBD) and define its binding site for the N-terminal portion of the VP16 transactivation domain (TADn). A hydrophobic furrow, formed by a β-barrel and two α-helices in MED25 VBD, interacts tightly with VP16 TADn. Mutations in this furrow prevent binding of VP16 TAD to MED25 VBD and interfere with the ability of overexpressed MED25 VBD to inhibit VP16-dependent transcriptional activation in vivo. This detailed molecular understanding of transactivation by the benchmark activator VP16 could provide important insights into viral and cellular gene activation mechanisms. View full text Figures at a glance * Figure 1: VP16-activated transcription. VP16, OCT-1 and HCF-1 form a VP16-induced complex33. The VP16 transactivation domain (TAD) interacts with the MED25VP16 binding domain (VBD) to recruit the Mediator and activate transcription. * Figure 2: Solution structure of the MED25 VBD determined by NMR. () The 25 lowest-energy structures are shown overlaid using the secondary structure elements in side-by-side stereoview. ( Cartoon drawing of MED25 VBD; the seven β-strands forming the barrel and the three α-helices are depicted in red and blue, respectively. The long α3 helix docks on the barrel by making close contact with β5, β6 and α1. Helix α2 caps the barrel from one side. A color-coded electrostatic surface potential shows a negative patch surrounded by areas of positive potential with a hydrophobic furrow in the center (, middle panel), highlighted by arrows. * Figure 3: MED25 VBD adopts a rare seven-strand β-barrel fold. Cartoon drawing of MED25 VBD accompanied by three structural homologs found using DALI34: two β-barrel-domains from the KU70–KU80 complex (PDB 1JEQ26) and the SPOC (PDB 1OW1)25 domain of SPEN. The three homologous structures have a different topology and lack the C-terminal helix present in MED25 VBD. The three MED25 VBD homologs are shown from left to right in decreasing degree of structural homology. * Figure 4: Interaction of VP16 TADn with MED25 VBD. () Overlay of 1H-15N HSQC spectra of VP16 TADn alone (blue) and in the presence of 1.3 equivalents of unlabeled MED25 VBD (red). Although the NMR signals of MED25 VBD in the bound state (, red signals) still show reasonable line-width and intensity, the signals of the MED25-binding site of VP16 TADn show extensive chemical exchange line broadening and attenuation, in particular for far-shifted signals (circled peaks with assignment indicated). () Overlay of 1H-15N HSQC spectra of MED25 VBD alone (blue) and in the presence of 1.3 equivalents of unlabeled VP16 TADn (red). () Changes in the chemical shifts of the amide proton and nitrogen atoms of VP16 TADn upon binding of MED25 VBD were plotted against the residue number. Amide resonances circled in are highlighted by arrows. () Changes in the chemical shifts of the amide proton and nitrogen atoms of MED25 VBD upon binding of VP16 TADn were plotted against the residue number. Residues wherein amide signals underwent changes in! chemical shift of greater than 0.4 p.p.m. are highlighted by arrows. () A cartoon representation of MED25 VBD shows the clustering of the residues undergoing chemical shifts changes greater than 0.4 p.p.m. (drawn with space-filling spheres) upon interaction with VP16 TADn in or near the hydrophobic furrow. The changes are clustered on β3, β5, β6 and α3. () Residues Ile453, Leu458, ALa495, Cys497, Val510, Met512, Phe533, Ile537 and Ile541 of MED25 VBD form a central hydrophobic pocket with in the large hydrophobic furrow. Several residues in or near this pocket showed the most pronounced chemical shift changes upon binding of VP16 TADn (Fig. 4e). * Figure 5: Functional studies of mutant MED25 VBD. () VP16 full-length TAD pulldown assays with MED25 VBD mutants. K447E, Q451E, H499E and K545E-mutated MED25 VBD shows weak or no binding to VP16 TAD (marked with an arrow). () Whereas wild-type MED25 VBD acts in a dominant negative fashion to inhibit Gal4p-VP16 full-length TAD-dependent transcription, the K447E, Q451E, H499E and K545E-mutated MED25 VBDs are incapable of inhibiting VP16-mediated transcription (WT: P < 0.01, t-test, one tailed, error bars represent s.d.). () The MED25 VBD Q451E mutation on β3 adjacent to the hydrophobic pocket disrupts binding of VP16 TADn to MED25 VBD. 1H-15N HSQC spectra of free VP16 TADn (left), at 1:1.3 excess of wild-type MED25 VBD (middle) and with 1:1.5 excess of Q451E MED25 VBD (right) show that VP16 TADn binds only loosely—as seen by minor chemical shift changes—to mutant MED25 VBD, without adopting a folded conformation. All far-shifted signals caused by the addition of wild-type MED25 VBD (middle, circled in red with assignment! indicated) are missing when the mutant MED25 VBD is added to 15N-labeled VP16 TADn (right). Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 2KY6 * 2KY6 Referenced accessions GenBank * 17139 Protein Data Bank * 1OW1 * 1JEQ * 1OW1 * 1JEQ Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Madhura Kulkarni & * Tingfang Yi Affiliations * Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA. * Alexander G Milbradt, * Tingfang Yi, * Koh Takeuchi, * Zhen-Yu J Sun, * Rafael E Luna, * Philipp Selenko & * Gerhard Wagner * Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts, USA. * Madhura Kulkarni & * Anders M Näär * Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA. * Madhura Kulkarni & * Anders M Näär * Present addresses: Biomedical Information Research Center, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan (K.T.), and Department of NMR-assisted Structural Biology, Leibniz Institute of Molecular Pharmacology, Berlin, Germany (P.S.). * Koh Takeuchi & * Philipp Selenko Contributions A.G.M. prepared protein samples, recorded and analyzed NMR data, calculated the structure, designed the MED25 VBD mutants and co-wrote the paper; M.K. generated point mutations, did pulldown assays and assisted with transcription assays; T.Y. did transcription assays; R.E.L. assisted with transcription assays, data interpretation and writing of the paper; K.T. and P.S. cloned the original construct, collected initial NMR data, obtained preliminary assignment, and assisted with editing and writing the paper; Z.-Y.J.S. assisted in recording NMR data, data analysis and structure calculation; A.M.N. and G.W. initiated the project, helped design experiments, advised on data collection and interpretation, and participated in writing and editing the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Anders M Näär or * Gerhard Wagner Author Details * Alexander G Milbradt Search for this author in: * NPG journals * PubMed * Google Scholar * Madhura Kulkarni Search for this author in: * NPG journals * PubMed * Google Scholar * Tingfang Yi Search for this author in: * NPG journals * PubMed * Google Scholar * Koh Takeuchi Search for this author in: * NPG journals * PubMed * Google Scholar * Zhen-Yu J Sun Search for this author in: * NPG journals * PubMed * Google Scholar * Rafael E Luna Search for this author in: * NPG journals * PubMed * Google Scholar * Philipp Selenko Search for this author in: * NPG journals * PubMed * Google Scholar * Anders M Näär Contact Anders M Näär Search for this author in: * NPG journals * PubMed * Google Scholar * Gerhard Wagner Contact Gerhard Wagner 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 (9M) Supplementary Figures 1–9 and Supplementary Table 1 Additional data
• Apolipoprotein A-I structural organization in high-density lipoproteins isolated from human plasma
  - Nat Struct Mol Biol 18(4):416-422 (2011)
  Nature Structural & Molecular Biology | Article Apolipoprotein A-I structural organization in high-density lipoproteins isolated from human plasma * Rong Huang1 * R A Gangani D Silva1 * W Gray Jerome2 * Anatol Kontush3, 4, 5 * M John Chapman3, 4, 5 * Linda K Curtiss6 * Timothy J Hodges7 * W Sean Davidson1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:416–422Year published:(2011)DOI:doi:10.1038/nsmb.2028Received30 June 2010Accepted15 December 2010Published online13 March 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 High-density lipoproteins (HDLs) mediate cholesterol transport and protection from cardiovascular disease. Although synthetic HDLs have been studied for 30 years, the structures of human plasma–derived HDL and its major protein apolipoprotein apoA-I are unknown. We separated normal human HDL into five density subfractions and then further isolated those containing predominantly apoA-I (LpA-I). Using cross-linking chemistry and mass spectrometry, we found that apoA-I adopts a structural framework in these particles that closely mirrors that in synthetic HDL. We adapted established structures for synthetic HDL to generate the first detailed models of authentic human plasma HDL in which apoA-I adopts a symmetrical cage-like structure. The models suggest that HDL particle size is modulated by means of a twisting motion of the resident apoA-I molecules. This understanding offers insights into how apoA-I structure modulates HDL function and its interactions with other apolipopro! teins. View full text Figures at a glance * Figure 1: Isolation and characterization of human plasma LpA-I HDL particles. () An 18% SDS-PAGE analysis of the density-isolated HDL particles before (denoted as HDL2b–3c) and after (denoted as LpA-I2b–3c) sulfhydryl covalent chromatography. Lipid-free apoA-I is shown in lane 2 as a reference. Alb., albumin. () The indicated LpA-I subfractions were analyzed on a calibrated Superdex 200 gel-filtration column. The figure shows a representative result from three analyses of three independently prepared samples. () An 8–25% native PAGE analysis of BS3 cross-linked LpA-I subfractions. See Table 1 for calculated diameters from panels and . All gels were stained with Coomassie blue. * Figure 2: Estimation of the number of apoA-I molecules per particle in LpA-I subfractions. () A 4–15% SDS-PAGE analysis of unmodified (−) and cross-linked (×) LpA-I subfractions. The gel was stained with Coomassie blue. () Predicted diameters for the LpA-I2b particle given the experimentally derived particle compositions calculated at various numbers of apoA-I molecules per particle (see text). The dashed line shows the experimental particle diameter (averaged from gel filtration and native PAGE, Table 1) for this particle. The bracket indicates the range of possible apoA-I molecules per particle determined by SDS-PAGE analysis of cross-linked LpA-I2b particles (for example, lane 3 in panel ). * Figure 3: Investigating apoA-I conformation in LpA-I particles. () Far UV circular dichroism spectra for the indicated LpA-I subfractions. The inset shows the calculated percent helicity of apoA-I with a typical s.d. of ± 5%. () An 8–25% SDS-PAGE analysis of LpA-I subfractions subjected to limited trypsin digestion and stained with Coomassie blue. Both panels show representative results from two independent experiments. * Figure 4: Trefoil model of apoA-I on spherical particles with experimental cross-links derived from human plasma HDL particles. The model contains three apoA-I molecules (modeled with residues 40–243) shown as ribbons, each in a different color. For clarity, no lipids are shown. The same model is shown from two views: (left) looking from the intersection of helix 5 (residue 133) in each molecule and (right) looking down from helix 10 (residue 233). Cross-links derived from physiological HDL in Table 2 judged to fit the 5/5 form of the double-belt/trefoil model (see text) are shown as red lines connecting the α-carbons of the involved lysine residues (shown as spheres). Lysines were considered plausible if the α-carbons were within 25 Å (11.4 Å for BS3 and 6.8 Å for each lysine side chain). Clearly visible cross-links are labeled with a letter and refer to: (A) 133-118, (B) 133-140, (C) 140-118, (D) 107-118, (E) 96-106, (F) 88-94, (G) 88-96, (H) 94-96, (I) 208-59, (J) 195-77, (K) 40-239, (L) 226-239, (M) 239-208, (N) 40-239, (O) 239-239 and (P) 96-118. Additional molecules of apoA-I can be adde! d to the trefoil as illustrated in Figure 5 without affecting the cross-linking patterns, but they are not shown for clarity. * Figure 5: Incorporation of additional apoA-I molecules to the trefoil model and apoA-I adaptation to smaller particle diameters. () Schematic representation of the three-molecule trefoil model as originally proposed, with each molecule of apoA-I shown in a different color (see Fig. 4 for more detail). The lighter color band on each molecule represents the N terminus (residue 44, as the model was built in the absence of residues 1–43). The inset is a schematic top view showing the bend angles of each apoA-I. () Pentameric complex proposed for the structure of LpA-I2b. () An idealized, fully extended tetrameric complex. () Twisted tetrameric complex with a reduced particle diameter as proposed for LpA-I2a. * Figure 6: Molecular twist required to attain the experimentally LpA-I particle diameters. The helical domains of apoA-I molecules are represented as tubes extending across the particle surface with each molecule in a different color. In LpA-I2b, apoA-I helical domains from five molecules are all maximally extended. The smaller particles contain four apoA-I molecules per particle and each pole has been twisted by an angle ΘΘ such that the complex diameter (D) matches the experimentally determined values (average of the diameter values from gel filtration and native PAGE in Table 1). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio, USA. * Rong Huang, * R A Gangani D Silva & * W Sean Davidson * Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee, USA. * W Gray Jerome * Université Pierre et Marie Curie-Paris 6, Paris, France. * Anatol Kontush & * M John Chapman * National Institute of Health and Medical Research, Dyslipoproteinemia and Atherosclerosis Research Unit, Paris, France. * Anatol Kontush & * M John Chapman * Assistance Publique–Hopitaux de Paris, Groupe hospitalier Pitié-Salpétrière, Paris, France. * Anatol Kontush & * M John Chapman * Department of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California, USA. * Linda K Curtiss * Department of Mathematical Sciences, University of Cincinnati, Cincinnati, Ohio, USA. * Timothy J Hodges Contributions R.H., M.J.C. and W.S.D. designed the research plan. R.H., R.A.G.D.S., L.K.C., W.G.J. and A.K. conducted experiments. R.H., W.S.D. and T.J.H. analyzed data, and R.H. and W.S.D. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * W Sean Davidson Author Details * Rong Huang Search for this author in: * NPG journals * PubMed * Google Scholar * R A Gangani D Silva Search for this author in: * NPG journals * PubMed * Google Scholar * W Gray Jerome Search for this author in: * NPG journals * PubMed * Google Scholar * Anatol Kontush Search for this author in: * NPG journals * PubMed * Google Scholar * M John Chapman Search for this author in: * NPG journals * PubMed * Google Scholar * Linda K Curtiss Search for this author in: * NPG journals * PubMed * Google Scholar * Timothy J Hodges Search for this author in: * NPG journals * PubMed * Google Scholar * W Sean Davidson Contact W Sean Davidson 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 and 2, and Supplementary Methods Additional data
• ABC ATPase signature helices in Rad50 link nucleotide state to Mre11 interface for DNA repair
  - Nat Struct Mol Biol 18(4):423-431 (2011)
  Nature Structural & Molecular Biology | Article ABC ATPase signature helices in Rad50 link nucleotide state to Mre11 interface for DNA repair * Gareth J Williams1, 7 * R Scott Williams2, 3, 6, 7 * Jessica S Williams2, 6, 7 * Gabriel Moncalian2, 3, 6, 7 * Andrew S Arvai2, 3 * Oliver Limbo2 * Grant Guenther2, 3 * Soumita SilDas1 * Michal Hammel4 * Paul Russell2, 5 * John A Tainer1, 2, 3 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:423–431Year published:(2011)DOI:doi:10.1038/nsmb.2038Received22 November 2010Accepted15 February 2011Published online27 March 2011Corrected online06 April 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 The Rad50 ABC–ATPase complex with Mre11 nuclease is essential for dsDNA break repair, telomere maintenance and ataxia telangiectasia–mutated kinase checkpoint signaling. How Rad50 affects Mre11 functions and how ABC–ATPases communicate nucleotide binding and ligand states across long distances and among protein partners are questions that have remained obscure. Here, structures of Mre11–Rad50 complexes define the Mre11 2-helix Rad50 binding domain (RBD) that forms a four-helix interface with Rad50 coiled coils adjoining the ATPase core. Newly identified effector and basic-switch helix motifs extend the ABC–ATPase signature motif to link ATP-driven Rad50 movements to coiled coils binding Mre11, implying an ~30-Å pull on the linker to the nuclease domain. Both RBD and basic-switch mutations cause clastogen sensitivity. Our new results characterize flexible ATP-dependent Mre11 regulation, defects in cancer-linked RBD mutations, conserved superfamily basic switches an! d motifs effecting ATP-driven conformational change, and they provide a unified comprehension of ABC–ATPase activities. View full text Figures at a glance * Figure 1: The Mre11RBD–Rad50 interface. () pfRad50 and pfMre11 construct schematics for domain mapping and crystallizations. pfRad50-link constructs contain Gly-Ser repeat sequences to intramolecularly link Rad50 N and C lobes. () Mapping of Mre11 RBD. Top, His6-tagged Rad50 was coexpressed with Mre11 variants (I–V) shown in . Bottom, His6-tagged Mre11 variants (V–VIII) were coexpressed with untagged pfRad50-link1. Minimal pfMre11 polypeptide (Mre11RBD, residues 348–381) interacts with Rad50-link1. MW, molecular weight. () Sequence alignment of the linker region connecting the Mre11 RBD to the nuclease-capping domain in pfMre11, S. cerevisiae (scMre11), S. pombe (spMre11), Xenopus laevis (xlMre11) and human (hsMre11). Shaded regions show well-conserved residues. Disordered residues are shown in red, as seen in pfMre11 crystal structures or as predicted by Disopred2. () Mre11RBD–Rad50 interface, shown in orthogonal stereo views. The hydrophobic Mre11RBD–Rad50 interaction core is augmented by four flanking! complementary salt-bridge interactions, with acidic residues from Mre11 RBD interacting with four positively charged Rad50 surface residues. () Superimposition of two nucleotide-free Mre11RBD–Rad50 crystal forms. The core Mre11RBD–Rad50 interface is maintained, but an ~45° rotation about the base of the Rad50 coiled coil identifies a flexible linkage to Rad50 ATPase domains. Residues equivalent to those mutated in rad50S yeast phenotypes are shown in space-fill representations. * Figure 2: A conserved interface links eukaryotic Mre11 and Rad50. () Multiple sequence alignment of Mre11 RBD from pfMre11, scMre11, spMre11, xlMre11 and hsMre11. Shaded regions show well-conserved residues. CL→RR and CV→RR (highlighted by red boxes) mark hydrophobic to charged surface substitutions introduced into S. pombeMre11 RBD. HsMre11 mutations identified in somatic colorectal cancers are highlighted (solid red circle, point mutation; triangle, truncation7). () S. pombeMre11 RBD variant interactions with Rad50, Nbs1 and the Mre11 homodimeric interaction analyzed by two-hybrid. Growth on Dex-WL plates (minimal glucose medium lacking tryptophan and leucine) indicates the reporter strain transformed with plasmids pGADT7 (Gal4 activating domain) and pGBKT7 (Gal4 DNA binding domain) fused to the respective proteins. Growth on Dex-LWH (less stringent; lacking histidine) and Dex-LWHA (more stringent; lacking histidine and adenine) indicates a positive two-hybrid interaction. We have previously shown that pGBKT7-mre11-WT alone does not ! autoactivate9. Mre11 RBD mutants fail to interact with Rad50 yet retain homodimerization and Nbs1 interactions. Strains used are detailed in Supplementary Table 1. * Figure 3: The Mre11–Rad50 interaction interface coordinates DSBR in S. pombe. () Expression levels of Myc-tagged Mre11 variants. () Mre11 RBD variant sensitivity to UV, hydroxyurea (HU) and CPT. () Mre11 RBD variants are ionizing radiation (IR) sensitive. This plot is representative of two independent experiments (Supplementary Fig. 2). () The IR, UV, HU and CPT survival defects of mre11-RRRR are suppressed by Ku80 elimination. This rescue depends on Exo1. Strains used are detailed in Supplementary Table 1. * Figure 4: The M2R2-head assembly. () SAXS analysis of the M2R2 head reveals a transition from a conformationally flexible, open complex to a globular, closed complex upon ATP binding. Left, experimental SAXS curves of the M2R2 head without (−ATP) and with (+ATP) nucleotide. Fits of single and MES models of M2R2 heads to −ATP (middle panel) and +ATP (right panel) data. Models are shown as surfaces with Mre11 core dimer colored black and Rad50 domains with attached Mre11 RBD colored for open (magenta), partially open (blue), closed (green) and ATP-bound (red) conformations. Fits to the experimental data are shown for single models (dashed line) and the MES ensemble (cyan line) with quality of fit shown by χ2. () Mre11RBD–Rad50-link1–AMP:PNP–Mg2+ complex architecture. Left, Mre11RBD (green) binds to Rad50 coiled-coil base. Right, schematic of the structure. () Orthogonal views of the complex as in . See Table 1 for data processing and refinement statistics. * Figure 5: Rad50 nucleotide-binding induced conformational changes. () Nucleotide free (−AMP:PNP) and bound (+AMP:PNP) Rad50 conformations. Nucleotide binding is coordinated by the signature motif, Q loop, P loop, and induces an ~35° subdomain rotation. Rad50 N lobe rotation drives the π-helix wedge into the signature-coupling helices, dramatically altering signature-coupling helix conformation relative to ATPase subdomain interactions. Motifs are colored as in key. () Twenty salt-bridge switches rearrange upon nucleotide binding and coordinate domain rotations (see Supplementary Movie 1). Blue (positive) and red (negative) circles highlight charged residues. The Mre11 RBD is highlighted by a green surface representation of Rad50 residues involved in the interface. () Signature helix Arg797 and Arg805 rearrangements link nucleotide binding with domain rotations, conformational change of the signature-coupling helices and Q loop, and motions in the Rad50 coiled coils (see Supplementary Movie 2). The Mre11 RBD is highlighted as in . () Nuc! leotide-binding induced Rad50 ATPase C-lobe rotation relative to the N lobe drives coiled-coil repositioning to affect bound Mre11 RBD, highlighted as in . The Rad50 domain rotation is transduced through coiled-coil repositioning (see Supplementary Movie 3), into a linear pull on the linker between the Mre11 RBD and nuclease-capping domain as depicted by dashed arrows. * Figure 6: ABC–ATPase superfamily conserved basic-switch residues in Rad50 coordinate DSBR in S. pombe. () Rad50 sequence alignment with ABC transporters shows the extended signature motif, with well-conserved residues shaded. Red circles (point mutations) and triangles (truncations) denote cystic fibrosis causing CFTR mutations. () S. pombeRad50 basic-switch variants on the signature helix are defective for DSBR. Left: Expression levels of TAP-tagged Rad50 variants, as probed by PAP antibody. Right: ionizing radiation (IR), CPT, hydroxyurea (HU) and UV sensitivity of Rad50 basic-switch variants. Strains used are detailed in Supplementary Table 1. * Figure 7: Topologically equivalent signature helices connect nucleotide binding to conformational changes in the ABC-ATPase superfamily. Rad50 ABC-ATPase molecular surface with attached coiled coil and mapped Mre11 RBD compared to MalK ABC-ATPase with interacting MalF transmembrane protein. The extended signature helix (purple) and signature-coupling helices or helix (cyan) connect nucleotide binding to movements of attached functional domains and proteins. Accession codes * Abstract * Accession codes * Change history * Author information * Supplementary information Primary accessions Protein Data Bank * 3QKS * 3QKR * 3QKU * 3QKT * 3QKS * 3QKR * 3QKU * 3QKT Referenced accessions Protein Data Bank * 3DSC * 3DSD * 1II7 * 3DSC * 3DSD * 1II7 Change history * Abstract * Accession codes * Change history * Author information * Supplementary informationErratum 06 April 2011In the version of this article initially published, the acronym ENIGMA was spelled out incorrectly in the Acknowledgments section. The error has been corrected in the HTML and PDF versions of the article. Author information * Abstract * Accession codes * Change history * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Gareth J Williams, * R Scott Williams, * Jessica S Williams & * Gabriel Moncalian Affiliations * Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. * Gareth J Williams, * Soumita SilDas & * John A Tainer * Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA. * R Scott Williams, * Jessica S Williams, * Gabriel Moncalian, * Andrew S Arvai, * Oliver Limbo, * Grant Guenther, * Paul Russell & * John A Tainer * Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA. * R Scott Williams, * Gabriel Moncalian, * Andrew S Arvai, * Grant Guenther & * John A Tainer * Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. * Michal Hammel * Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA. * Paul Russell * Present addresses: Laboratory of Structural Biology, National Institute of Environmental Health Sciences, US National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA (R.S.W. and J.S.W.) and Instituto de Biomedicina y Biotecnología de Cantabria, Santander, Spain (G.M). * R Scott Williams, * Jessica S Williams & * Gabriel Moncalian Contributions G.J.W. analyzed results, did SAXS experiments and wrote the manuscript. J.S.W. and O.L. did S. pombe experiments and analysis. G.M. and R.S.W. solved crystal structures. A.S.A. refined structures. S.S. and G.G. purified proteins. M.H. assisted with SAXS analyses. R.S.W., J.S.W., P.R. and J.A.T. designed research, analyzed results and helped write the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * R Scott Williams or * Paul Russell or * John A Tainer Author Details * Gareth J Williams Search for this author in: * NPG journals * PubMed * Google Scholar * R Scott Williams Contact R Scott Williams Search for this author in: * NPG journals * PubMed * Google Scholar * Jessica S Williams Search for this author in: * NPG journals * PubMed * Google Scholar * Gabriel Moncalian Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew S Arvai Search for this author in: * NPG journals * PubMed * Google Scholar * Oliver Limbo Search for this author in: * NPG journals * PubMed * Google Scholar * Grant Guenther Search for this author in: * NPG journals * PubMed * Google Scholar * Soumita SilDas Search for this author in: * NPG journals * PubMed * Google Scholar * Michal Hammel Search for this author in: * NPG journals * PubMed * Google Scholar * Paul Russell Contact Paul Russell Search for this author in: * NPG journals * PubMed * Google Scholar * John A Tainer Contact John A Tainer Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Change history * Author information * Supplementary information Movies * Supplementary Movie 1 (8M) Nucleotide-induced Rad50 conformational changes. Rad50 structures with coiled-coil regions in the absence (start position) and presence (end position) of nucleotide were superimposed and movies generated by morphing between states in PyMOL (DeLano Scientific LLC, Palo Alto, CA, U.S.A. http://www.pymol.org). Supplementary Movie 1 shows the N-lobe rotation with respect to the C-lobe. Supplementary Movie 2 shows the C-lobe rotation with respect to the N-lobe. Supplementary Movie 3 shows the C-lobe rotation with respect to the N-lobe from the side. The extended signature motif (magenta) and signature coupling helices (cyan) are highlighted. Residues in the text are shown as sticks with the extended signature motif basic-switch residues (Arg797 and Arg805) highlighted by representation of side chain nitrogens as spheres. To relate the movements to nucleotide binding AMP:PNP is shown as sticks, although it is only observed in the structure of the nucleotide bound form. To relate t! he movements to bound Mre11 RBD, residues on the Rad50 coiled-coils involves in the Mre11RBD–Rad50 interface are shown in a green transparent surface representation. For clarity only one Rad50 molecule is shown, although in structures in the presence of nucleotide dimerization occurs with AMP:PNP sandwiched between ABC-ATPase domains. * Supplementary Movie 2 (9M) Nucleotide-induced Rad50 conformational changes. Rad50 structures with coiled-coil regions in the absence (start position) and presence (end position) of nucleotide were superimposed and movies generated by morphing between states in PyMOL (DeLano Scientific LLC, Palo Alto, CA, U.S.A. http://www.pymol.org). Supplementary Movie 1 shows the N-lobe rotation with respect to the C-lobe. Supplementary Movie 2 shows the C-lobe rotation with respect to the N-lobe. Supplementary Movie 3 shows the C-lobe rotation with respect to the N-lobe from the side. The extended signature motif (magenta) and signature coupling helices (cyan) are highlighted. Residues in the text are shown as sticks with the extended signature motif basic-switch residues (Arg797 and Arg805) highlighted by representation of side chain nitrogens as spheres. To relate the movements to nucleotide binding AMP:PNP is shown as sticks, although it is only observed in the structure of the nucleotide bound form. To relate t! he movements to bound Mre11 RBD, residues on the Rad50 coiled-coils involves in the Mre11RBD–Rad50 interface are shown in a green transparent surface representation. For clarity only one Rad50 molecule is shown, although in structures in the presence of nucleotide dimerization occurs with AMP:PNP sandwiched between ABC-ATPase domains * Supplementary Movie 3 (7M) Nucleotide-induced Rad50 conformational changes. Rad50 structures with coiled-coil regions in the absence (start position) and presence (end position) of nucleotide were superimposed and movies generated by morphing between states in PyMOL (DeLano Scientific LLC, Palo Alto, CA, U.S.A. http://www.pymol.org). Supplementary Movie 1 shows the N-lobe rotation with respect to the C-lobe. Supplementary Movie 2 shows the C-lobe rotation with respect to the N-lobe. Supplementary Movie 3 shows the C-lobe rotation with respect to the N-lobe from the side. The extended signature motif (magenta) and signature coupling helices (cyan) are highlighted. Residues in the text are shown as sticks with the extended signature motif basic-switch residues (Arg797 and Arg805) highlighted by representation of side chain nitrogens as spheres. To relate the movements to nucleotide binding AMP:PNP is shown as sticks, although it is only observed in the structure of the nucleotide bound form. To relate t! he movements to bound Mre11 RBD, residues on the Rad50 coiled-coils involves in the Mre11RBD–Rad50 interface are shown in a green transparent surface representation. For clarity only one Rad50 molecule is shown, although in structures in the presence of nucleotide dimerization occurs with AMP:PNP sandwiched between ABC-ATPase domains PDF files * Supplementary Text and Figures (8M) Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Methods Additional data
• How mutations in tRNA distant from the anticodon affect the fidelity of decoding
  - Nat Struct Mol Biol 18(4):432-436 (2011)
  Nature Structural & Molecular Biology | Article How mutations in tRNA distant from the anticodon affect the fidelity of decoding * T Martin Schmeing1, 2, 3 * Rebecca M Voorhees1, 3 * Ann C Kelley1 * V Ramakrishnan1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:432–436Year published:(2011)DOI:doi:10.1038/nsmb.2003Received19 October 2010Accepted08 December 2010Published online06 March 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 ribosome converts genetic information into protein by selecting aminoacyl tRNAs whose anticodons base-pair to an mRNA codon. Mutations in the tRNA body can perturb this process and affect fidelity. The Hirsh suppressor is a well-studied tRNATrp harboring a G24A mutation that allows readthrough of UGA stop codons. Here we present crystal structures of the 70S ribosome complexed with EF-Tu and aminoacyl tRNA (native tRNATrp, G24A tRNATrp or the miscoding A9C tRNATrp) bound to cognate UGG or near-cognate UGA codons, determined at 3.2-Å resolution. The A9C and G24A mutations lead to miscoding by facilitating the distortion of tRNA required for decoding. A9C accomplishes this by increasing tRNA flexibility, whereas G24A allows the formation of an additional hydrogen bond that stabilizes the distortion. Our results also suggest that each native tRNA will adopt a unique conformation when delivered to the ribosome that allows accurate decoding. View full text Figures at a glance * Figure 1: Overview of miscoding mutations and Trp-tRNATrp bound in the A/T state. () The traditional cloverleaf diagram of Trp-tRNATrp showing the locations of the miscoding mutations A9C (orange) and G24A (red). () When bound to the ribosome along with EF-Tu, the aminoacyl tRNA (green) adopts the distorted A/T conformation. Structures were also determined for Trp-tRNATrp containing mutations at position 24 (red spheres) and 9 (orange spheres). () The A/T conformation requires a ~30° bend in the tRNA body (green) compared to a canonical tRNA (gray)19. This bend is achieved through two isolated regions of distortion, first in the anticodon stem and the second in the D-stem, where both the A9C and G24A mutations are located. * Figure 2: Comparison of cognate and near-cognate structures in the decoding center. () Unbiased Fo – Fc electron density (showed at 1.3σ within 2 Å of EF-Tu) is shown for residues in the wobble position of the codon-anticodon helix. () Binding of a near-cognate tRNA (A9C on UGA, orange; G24A on UGA, red), compared to a cognate tRNA (G24A on UGG, yellow; tRNATrp on UGG, green) results in a shift in both the tRNA anticodon and mRNA codon. () The distortion in the anticodon caused by this mismatch is propagated three residues, resulting in a shift in the tRNA backbone for the G24A on UGA when compared to the G24A or tRNATrp on UGG. () This distortion does not, however, continue past residue 31; by residue 28, the backbone of the G24A on UGA has converged with that of the G24A on UGG. * Figure 3: Miscoding by the G24A and A9C Trp-tRNATrp. () Mutation of G24A (red) facilitates formation of a hydrogen bonding interaction that is not possible in native tRNATrp (green) between the exocylic amine of A24 and O6 of G44. This interaction is facilitated by a small shift in the RNA backbone between residues 20–30 and a commensurate movement between residues 9–16. () The interaction between residues 24 and 44 in the G24A tRNATrp is only possible because of a base pair between residues A26–U45 in tRNATrp that dictates the backbone conformation in this region. In contrast, tRNAThr (purple)14 contains two purines at these positions—G26 and G45—which push the tRNA backbone apart and separate residues G24 and A44. That the A26–U45 base pair is not conserved in bacterial tRNATrp further illustrates that evolution has fine-tuned each tRNA to find a unique solution to tRNA bending during decoding. () In native tRNATrp (shown in green), a base triple forms between residues 9:12:23, which is at the junction of three s! eparate strands of the tRNA. Mutation of residue 9 to a cytosine (shown in orange) weakens both the packing and hydrogen-bonding of this base-triple, which could result in higher flexibility of the tRNA body and explain its ability to miscode. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 2Y0U * 2Y0V * 2Y0W * 2Y0X * 2Y0Y * 2Y0Z * 2Y12 * 2Y13 * 2Y14 * 2Y15 * 2Y16 * 2Y17 * 2Y18 * 2Y19 * 2Y10 * 2Y11 * 2Y0U * 2Y0V * 2Y0W * 2Y0X * 2Y0Y * 2Y0Z * 2Y12 * 2Y13 * 2Y14 * 2Y15 * 2Y16 * 2Y17 * 2Y18 * 2Y19 * 2Y10 * 2Y11 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * T Martin Schmeing & * Rebecca M Voorhees Affiliations * MRC Laboratory of Molecular Biology, Cambridge, UK. * T Martin Schmeing, * Rebecca M Voorhees, * Ann C Kelley & * V Ramakrishnan * Present address: Biochemistry Department, McGill University, Montreal, Quebec, Canada. * T Martin Schmeing Contributions T.M.S. and R.M.V. designed the experiments, prepared and crystallized the ribosomal complexes, collected and processed X-ray crystallography data, determined, refined and analyzed the structures, and prepared the manuscript and figures. A.C.K. purified macromolecular components, prepared and crystallized ribosomal complexes and aided with collection of X-ray crystallography data. V.R. suggested the study and provided intellectual input and supervision. Competing financial interests V.R. is on the Senior Advisory Board of Rib-X Pharmaceuticals and both T.M.S. and V.R. hold stock options. Corresponding author Correspondence to: * T Martin Schmeing Author Details * T Martin Schmeing Contact T Martin Schmeing Search for this author in: * NPG journals * PubMed * Google Scholar * Rebecca M Voorhees Search for this author in: * NPG journals * PubMed * Google Scholar * Ann C Kelley Search for this author in: * NPG journals * PubMed * Google Scholar * V Ramakrishnan 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 1–3 Additional data
• Allosteric control of ligand-binding affinity using engineered conformation-specific effector proteins
  - Nat Struct Mol Biol 18(4):437-442 (2011)
  Nature Structural & Molecular Biology | Article Allosteric control of ligand-binding affinity using engineered conformation-specific effector proteins * Shahir S Rizk1 * Marcin Paduch1 * John H Heithaus1 * Erica M Duguid1 * Andrew Sandstrom1 * Anthony A Kossiakoff1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:437–442Year published:(2011)DOI:doi:10.1038/nsmb.2002Received14 September 2010Accepted07 December 2010Published online06 March 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 We describe a phage display methodology for engineering synthetic antigen binders (sABs) that recognize either the apo or the ligand-bound conformation of maltose-binding protein (MBP). sABs that preferentially recognize the maltose-bound form of MBP act as positive allosteric effectors by substantially increasing the affinity for maltose. A crystal structure of a sAB bound to the closed form of MBP reveals the basis for this allosteric effect. We show that sABs that recognize the bound form of MBP can rescue the function of a binding-deficient mutant by restoring its natural affinity for maltose. Furthermore, the sABs can enhance maltose binding in vivo, as they provide a growth advantage to bacteria under low-maltose conditions. The results demonstrate that structure-specific sABs can be engineered to dynamically control ligand-binding affinities by modulating the transition between different conformations. View full text Figures at a glance * Figure 1: Phage display selection strategy. MBP undergoes a conformational change through a hinge-bending motion upon binding to maltose (red). Carrying out the phage display selection without maltose generates sABs (yellow sphere) that bind preferentially to the open form of MBP, whereas selection in the presence of maltose leads to closed-specific sABs (green sphere). Placement of spheres indicates postulated binding modes of sABs to the different forms of MBP. * Figure 2: Influence of sABs on maltose binding. () The change in fluorescence of the MBP S233C Alexa 488 conjugate (solid line) upon addition of 1 mM maltose (dashed line). () Fluorescence maltose-binding curves of MBP S233C Alexa 488 without (•) or with 200 nM sAB MCS1 (⋄) or 200 nM sAB MCS4 (○). () Intrinsic tryptophan fluorescence of MBP without (solid line) or with 1 mM maltose (dashed line). () Fluorescence maltose-binding curves of MBP without (•) or with 200 nM sAB MOS1 (○). * Figure 3: Scatchard analysis of maltose binding. Data points from Figure 2 were replotted as r versus R / [maltose], where R is the fractional saturation of MBP with maltose. () Binding of maltose to MBP without sABs. () Maltose binding with 200 nM sAB MOS1. (,) Maltose binding with 200 nM sAB MCS1 () or 200 nM sAB MCS4 (). * Figure 4: Crystal structure of MBP–MCS2 complex. MBP (blue) is in the closed form, bound to one molecule of maltose (red). The sAB (heavy chain, green; light chain, yellow) interacts with MBP at the opposite side of the binding pocket, forming a wedge that favors the closed form. * Figure 5: The 'wedge' formed by the CDR loops of MCS2. () A group of bulky side chain residues within CDRH-3 of the sAB (green sticks) form the wedge structure, which interacts with a region within MBP (blue) that is only exposed in the closed, maltose-bound conformation. () Sequence of the CDR loops of MCS2. Bold letters, residues that interact with the MBP molecule. () Overlay of the MCS2–MBP complex with the open form of MBP (purple, PDB 1OMP12) indicates that the apo form of MBP clashes with the sAB CDR loops. * Figure 6: Rescuing binding function of an MBP mutant. The effect of the binding pocket mutation W62F on affinity of MBP for maltose was assessed using the emission change of Alexa 488 attached to Cys233. Binding affinity without sAB, and with either 200 nM sAB MCS4 or 200 nM sAB MCS1. The binding curve of MBP S233C with no binding pocket mutations (dashed line) is a reference. * Figure 7: Allosteric activity of sABs in vivo. Escherichia coli cells expressing sABs in the periplasm were grown in minimal media containing maltose as the sole carbon source. Cells expressing sAB MCS1 or sAB MCS4 and control cells expressing sAB-27, an actin-binding sAB14, versus maltose concentration. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3PGF * 3PGF Referenced accessions Protein Data Bank * 1OMP * 1OMP Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Biochemistry and Molecular Biology and the Institute for Biophysical Dynamics, University of Chicago, Chicago, Illinois, USA. * Shahir S Rizk, * Marcin Paduch, * John H Heithaus, * Erica M Duguid, * Andrew Sandstrom & * Anthony A Kossiakoff Contributions S.S.R. and A.A.K. designed the experiments and wrote the manuscript; S.S.R., M.P., J.H.H., E.M.D. and A.S. performed the experiments; E.M.D. solved the crystal structure. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Anthony A Kossiakoff Author Details * Shahir S Rizk Search for this author in: * NPG journals * PubMed * Google Scholar * Marcin Paduch Search for this author in: * NPG journals * PubMed * Google Scholar * John H Heithaus Search for this author in: * NPG journals * PubMed * Google Scholar * Erica M Duguid Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew Sandstrom Search for this author in: * NPG journals * PubMed * Google Scholar * Anthony A Kossiakoff Contact Anthony A Kossiakoff 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 (184K) Supplementary Figures 1 and 2 Additional data
• Molecular basis of purine-rich RNA recognition by the human SR-like protein Tra2-β1
  - Nat Struct Mol Biol 18(4):443-450 (2011)
  Nature Structural & Molecular Biology | Article Molecular basis of purine-rich RNA recognition by the human SR-like protein Tra2-β1 * Antoine Cléry1 * Sandrine Jayne1 * Natalya Benderska2 * Cyril Dominguez1 * Stefan Stamm2, 3 * Frédéric H-T Allain1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:443–450Year published:(2011)DOI:doi:10.1038/nsmb.2001Received26 February 2010Accepted06 December 2010Published online13 March 2011Corrected online25 March 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 Tra2-β1 is a unique splicing factor as its single RNA recognition motif (RRM) is located between two RS (arginine-serine) domains. To understand how this protein recognizes its RNA target, we solved the structure of Tra2-β1 RRM in complex with RNA. The central 5′-AGAA-3′ motif is specifically recognized by residues from the β-sheet of the RRM and by residues from both extremities flanking the RRM. The structure suggests that RNA binding by Tra2-β1 induces positioning of the two RS domains relative to one another. By testing the effect of Tra2-β1 and RNA mutations on the splicing of SMN2 exon 7, we validated the importance of the RNA-protein contacts observed in the structure for the function of Tra2-β1 and determined the functional sequence of Tra2-β1 in SMN2 exon 7. Finally, we propose a model for the assembly of multiple RNA binding proteins on this exon. View full text Figures at a glance * Figure 1: Study of the interaction between Tra2-β1 RRM and the 5′-AAGAAC-3′ RNA by ITC and NMR. () Schematic representation of the Tra2-β1 domains with linkers. The Tra2-β1 RRM is located between two RS domains. Amino acids delimitating each protein domain are indicated by their corresponding numbers according to the PDB sequence. The sequence of the recombinant protein used in this study is shown. Amino acids involved in the formation of β-strands and α-helices are red and blue, respectively. The conserved RNP1 and RNP2 motifs are indicated. () Kd determination of Tra2-β1 RRM in complex with the 5′-AAGAAC-3′ RNA by ITC. Kd is given ± s.d. () Superimposition of 1H-15N HSQC spectra representing NMR titration of the 15N-labeled Tra2-β1 RRM with increasing amounts of unlabeled 5′-AAGAAC-3′ RNA. Titration was performed at 40 °C in NMR buffer. The peaks corresponding to the free and RNA-bound protein states (RNA:protein ratios of 0.3:1 and 1:1) are blue, orange and red, respectively. Negative peaks corresponding to the amides of arginine side chains in the f! ree and RNA bound (1:1 ratio) states are green and orange, respectively. Black arrows indicate highest chemical shift perturbations (>0.9 p.p.m.) seen upon RNA binding. () Representation of the combined chemical shift perturbations (Δδ = [(δHN)2 + (δN/6.51)2]1/2) of Tra2-β1 RRM amides upon binding 5′-AAGAAC-3′ RNA as a function of the Tra2-β1 RRM amino acid sequence. The secondary structure elements of the RRM are shown at the top. The highest chemical shift perturbations (for Gly124, Ser194, Ile195 and Thr196) are indicated. * Figure 2: Overview of the solution structure of Tra2-β1 RRM in complex with the 5′-AAGAAC-3′ RNA. () Overlay of the 12 lowest-energy structures superimposed on the backbone of the protein-structured parts and heavy atoms of RNA. Protein backbone, gray; RNA heavy atoms, orange (phosphate atoms), yellow (C atoms), red (O atoms) and blue (N atoms). Only the ordered region of the RRM (residues 111–201) is shown. The N- and C-terminal regions of the RRM are shown in blue and orange, respectively. The N-terminal region of the protein has been truncated in the overlay on the left to avoid masking the RNA molecule. () Surface representation of the RRM (residues 106–201) and stick representation for the heavy atoms of the RNA molecule of the most representative structure of the complex. The protein surface is colored according to surface potential, with red and blue indicating negative and positive charges, respectively. The positively charged amino acids that are in contact with RNA are indicated. The RNA is colored as in . () Structure of the complex represented in ribbon (! protein backbone) and stick (RNA) representation. Color scheme as in . Important protein side chains involved in RNA interactions are represented as green sticks. The N- and C-terminal extremities are blue and orange, respectively. () Molecular recognition of the 5′-AGAA-3′ RNA sequence by Tra2-β1 RRM. Single structures focus on the intermolecular interactions that are most commonly observed in the structures. Protein-RNA interactions involving A2, G3, A4 and A5 are represented. Color schemes as in . Hydrogen bonds, purple dashed lines. Figures generated by MOLMOL49. * Figure 3: Effect of Tra2-β1 mutations on SMN2 exon 7 splicing. The SMN2 reporter minigene was cotransfected in HEK293 cells with increasing amounts (1 μg and 3 μg) of wild-type or mutant Tra2-β1 expression constructs. () Schematic representation of the SMN2 minigene. The exon 7 sequence is shown and the previously reported Tra2-β1 interacting sequence is underlined17. () Western blot experiment showing the expression of the GFP-tagged proteins after cell transfection with 1 and 3 μg of Tra2-β1 (wild type and mutant) expression constructs. β-actin was used as a loading control. () RT-PCR amplification of mRNAs produced from the SMN2 minigenes (wild type and mutants). The positions of PCR products with or without exon 7 are indicated on the right. The percentage of exon 7 inclusion in SMN2 transcripts was determined using ImageQuant. The negative control corresponds to the percentage of exon 7 inclusion in the absence of ectopic Tra2-β1 expression. () Statistical representation of at least three independent experiments showing the! strong negative effect of all the tested Tra2-β1 substitutions on SMN2 exon 7 inclusion. Student's test comparing Tra2-β1 constructs with the negative control; *P < 0.05, **P < 0.01, ***P < 0.001. Error bars are s.d. * Figure 4: Effect of nucleotide substitutions in the Tra2-β1 binding site on SMN2 exon 7 splicing. Wild-type and mutant SMN2 minigene plasmids (1 μg) were cotransfected with 3 μg of empty vector (–) or a vector expressing human Tra2-β1 (+). RT-PCR amplification of mRNAs produced from the SMN2 minigenes (wild type and mutants) is shown. The percentage of exon 7 inclusion in SMN2 transcripts was determined using ImageQuant. The positions of PCR products with or without exon 7 are indicated on the right. Lower panel, data from at least three independent experiments. Nucleotide substitutions are underlined. Error bars are s.d. * Figure 5: Binding of Tra2-β1 RRM might induce the positioning of the two RS domains on SMN exon 7 or the recruitment of hnRNP G and SRp30c. () Effect of deletion of RS1 or RS2 on SMN2 exon 7 splicing. Wild-type SMN2 minigene plasmid (1 μg) was cotransfected with 3 μg of empty vector (−) or a vector expressing wild-type, ΔRS1 or ΔRS2 Tra2-β1. RT-PCR amplification of mRNAs produced from the SMN2 minigene is shown. The positions of PCR products with or without exon 7 are indicated on the right. Right panel, results of at least three independent experiments. () Effect on SMN2 exon 7 splicing of UUU or CCA substitution (colored red and green, respectively) of the two CAA motifs located upstream of the Tra2-β1SMN exon 7 binding site in the absence or presence of hnRNP G or Tra2-β1 co-expression. Wild-type and mutant SMN2 minigene plasmids (1 μg) were cotransfected with 3 μg of empty vector (lanes 1–5) or a vector expressing wild-type hnRNP G (lanes 6–10) or Tra2-β1 (lanes 11–15). RT-PCR amplification of mRNAs produced from the SMN2 minigenes is shown. () Model of the assembly of Tra2-β1, hnRNP G and! SRp30c on SMN exon 7. Once the RRM of Tra2-β1 is bound to the RNA, the cross formed between the N and C termini might place RS domains near the RNA molecule, one upstream and the other downstream of the Tra2-β1 binding site. Our model proposes that the RS domains act as a platform to recruit hnRNP G and SRp30c on each side of the SMN exon 7 sequence bound by Tra2-β1. N-terminal (RS1) and C-terminal (RS2) RS domains are magenta and blue, respectively. The RNA is orange. The Tra2-β1 binding site and putative hnRNP G and SRp30c interacting sequences are red. Accession codes * Abstract * Accession codes * Change history * Author information * Supplementary information Primary accessions Protein Data Bank * 2KXN * 2KXN Referenced accessions GenBank * 16920 Change history * Abstract * Accession codes * Change history * Author information * Supplementary informationErratum 25 March 2011In the version of this article initially published online, in Figure 2c, the label "A1" in the right-hand panel was displaced to the left, and in the Results, "Tra2-β1" was incorrectly written as "Tra2-b1" in the subheading "Tra2-β1 binding to SMN might change position of RS domains." The errors have been corrected for all versions of this article. Author information * Abstract * Accession codes * Change history * Author information * Supplementary information Affiliations * Institute for Molecular Biology and Biophysics, Swiss Federal Institute of Technology, Zürich, Switzerland. * Antoine Cléry, * Sandrine Jayne, * Cyril Dominguez & * Frédéric H-T Allain * Institute of Biochemistry, University of Erlangen-Nürnberg, Erlangen, Germany. * Natalya Benderska & * Stefan Stamm * Department of Molecular and Cellular Biochemistry, Biomedical Biological Sciences Research Building, University of Kentucky, Lexington, Kentucky, USA. * Stefan Stamm Contributions F.H.-T.A. and S.S. designed the project; A.C. prepared protein and RNA samples for structural studies; A.C., C.D. and F.H.-T.A. analyzed NMR data; A.C. and C.D. did structure calculations; S.J., N.B. and A.C. did in vivo splicing assays; A.C. and C.D. did ITC measurements; N.B. conducted the coimmunoprecipitation experiments; all authors discussed the results and wrote and approved the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Frédéric H-T Allain Author Details * Antoine Cléry Search for this author in: * NPG journals * PubMed * Google Scholar * Sandrine Jayne Search for this author in: * NPG journals * PubMed * Google Scholar * Natalya Benderska Search for this author in: * NPG journals * PubMed * Google Scholar * Cyril Dominguez Search for this author in: * NPG journals * PubMed * Google Scholar * Stefan Stamm Search for this author in: * NPG journals * PubMed * Google Scholar * Frédéric H-T Allain Contact Frédéric H-T Allain 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 Methods Additional data
• Long telomeres are preferentially extended during recombination-mediated telomere maintenance
  - Nat Struct Mol Biol 18(4):451-456 (2011)
  Nature Structural & Molecular Biology | Article Long telomeres are preferentially extended during recombination-mediated telomere maintenance * Michael Chang1 * John C Dittmar2 * Rodney Rothstein1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:451–456Year published:(2011)DOI:doi:10.1038/nsmb.2034Received01 November 2010Accepted20 January 2011Published online27 March 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 Most human somatic cells do not express telomerase. Consequently, with each cell division their telomeres progressively shorten until replicative senescence is induced. Around 15% of human cancers maintain their telomeres using telomerase-independent, recombination-based mechanisms that are collectively termed 'alternative lengthening of telomeres' (ALT). In the yeast Saccharomyces cerevisiae, ALT cells are referred to as 'survivors'. One type of survivor (type II) resembles human ALT cells in that both are defined by the amplification of telomeric repeats. We analyzed recombination-mediated telomere extension events at individual telomeres in telomerase-negative yeast during the formation of type II survivors and found that long telomeres were preferentially extended. Furthermore, senescent cells with long telomeres were more efficient at bypassing senescence by the type II pathway. We speculate that telomere length may be important in determining whether cancer cells use t! elomerase or ALT to bypass replicative senescence. View full text Figures at a glance * Figure 1: STEX analysis of type II survivor formation. () Senescence was monitored in liquid culture by serially passaging four est2Δ spore products derived from an est2Δ/+ heterozygous diploid. (–) Analysis of sequenced VI-R telomeres from three est2Δ strains at days 5, 6 and 7: est2Δ no. 1 (), est2Δ no. 2 () and est2Δ no. 3 (). Analysis of telomeres from est2Δ no. 4 is shown in Supplementary Figure 1. Each bar represents an individual VI-R telomere and bars are sorted by the length of the undiverged sequence. The black portion of each bar represents the undiverged or unextended region of the telomere. The light gray portion represents the diverged or extended region of the telomere. For each est2Δ strain, the longest telomere without divergent sequence (hashed bar) is used as a reference telomere to which all other telomeres are compared to determine whether divergence has occurred. * Figure 2: Long telomeres are preferentially extended during type II survivor formation. () Sequenced VI-R telomeres from samples from pre-survivor cells (est2Δ no. 1, day 5; est2Δ no. 2, days 5 and 6; est2Δ no. 3, days 5 and 6; est2Δ no. 4, days 5 and 6) pooled and analyzed together. Bar below (in and ) indicates a run of telomeres with a significant increase in telomeres that show divergence, showing number of extended telomeres and total number of telomeres in the run as well as P value (determined by scan statistics; see Online Methods). () Sequenced telomeres from all samples where >30% but <70% of the telomeres show sequence divergence (est2Δ no. 1, day 6, telomere VI-R; est2Δ no. 3, day 7, telomere VI-R; est2Δ no. 4, day 7, telomeres VI-R and XIV-R) pooled and analyzed together. () Long unextended telomeres from day 7 samples can be explained by one of two models, both involving the preferential, recombination-mediated extension of long telomeres. Top, est2Δ no. 3 data from Figure 1d. The orange line highlights the observation that a number of tel! omeres from the day 7 sample contain undiverged regions that are longer than the longest day 6 telomere. Scenario A: long telomeres from day 7 (blue bars, right) originated from a long telomere from day 6 (blue bar indicated by blue arrow) that was not sequenced because such long telomeres were too rare in the day 6 sample to be detected in our assay. Scenario B: a long telomere from day 6 (red bar indicated by red arrow) was extended between days 6 and 7 and then duplicated several times, giving rise to the long telomeres in day 7 (red bars, right). See text. * Figure 3: Telomerase-negative rifΔ strains show accelerated senescence. () Telomere VI-R was sequenced from est2Δ rif1Δ, est2Δ rif2Δ and two different isolates of est2Δ rif1Δ rif2Δ after ~35 generations of clonal expansion following sporulation of an est2Δ/+ rif1Δ/+ rif2Δ/+ diploid. Sequenced telomeres were analyzed as in Figure 1. () Senescence rates were measured in liquid culture by serial passaging of est2Δ, est2Δ rif1Δ, est2Δ rif2Δ and est2Δ rif1Δ rif2Δ strains derived from the diploid used in . Mean ± s.e. for at least four independent spore isolates for each genotype are shown. * Figure 4: Telomere shortening rate is unchanged when RIF1 and RIF2 are deleted. () One isolate each of est2Δ and est2Δ rif1Δ rif2Δ was passaged as in Figure 3b. () At each passage, telomere lengths were determined by denaturing in-gel hybridization (see Online Methods). Vertical bar (right) indicates the position of the terminal restriction fragments of Y′ telomeres, which are present in more than half of yeast telomeres. Larger bands represent non-Y′-containing telomeres. A smear of telomeric signal appears in the est2Δ rif1Δ rif2Δ strain after 45 population doublings (PDs), which arise from type II survivors that have undergone telomere repeat amplification. () The telomere lengths from were quantified and plotted. * Figure 5: Model for the senescence of est2Δ and est2Δ rif1Δ rif2Δ strains. In an est2Δ strain (left), telomeres progressively shorten with each cell division. Short telomeres are uncapped and recruit DNA damage factors such as Rad52. The recruitment of DNA damage factors at one or a few shortened telomeres is insufficient to cause senescence until most or all telomeres have been sufficiently eroded. Bypass of senescence occurs by either the type I pathway (involving subtelomeric Y′ elements) or, less frequently, the type II pathway (involving the telomeric repeats). The type II pathway involves the preferential extension of long telomeres by recombination. In an est2Δ rif1Δ rif2Δ strain (right), telomeres become uncapped earlier and this leads to accelerated senescence. However, as the telomere shortening rate is unaffected by deletion of the RIF genes, telomeres are relatively long at senescence when compared to telomeres in an est2Δ strain. The longer telomeres recombine more efficiently, resulting in a marked increase in the fraction of e! st2Δ rif1Δ rif2Δ survivors that are type II. The Rad52 protein complex is depicted as a hexamer for simplicity. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Genetics and Development, Columbia University Medical Center, New York, New York, USA. * Michael Chang & * Rodney Rothstein * Department of Biological Sciences, Columbia University, New York, New York, USA. * John C Dittmar Contributions M.C. designed and carried out the experiments, analyzed the data and wrote the manuscript. J.C.D. did the statistical analysis of the STEX data. R.R. helped to analyze the data and write the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael Chang Author Details * Michael Chang Contact Michael Chang Search for this author in: * NPG journals * PubMed * Google Scholar * John C Dittmar Search for this author in: * NPG journals * PubMed * Google Scholar * Rodney Rothstein Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (532K) Supplementary Figure 1 and Supplementary Table 1 Additional data
• mRNA translocation occurs during the second step of ribosomal intersubunit rotation
  - Nat Struct Mol Biol 18(4):457-462 (2011)
  Nature Structural & Molecular Biology | Article mRNA translocation occurs during the second step of ribosomal intersubunit rotation * Dmitri N Ermolenko1, 2, 3 * Harry F Noller1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:457–462Year published:(2011)DOI:doi:10.1038/nsmb.2011Received09 June 2010Accepted15 December 2010Published online13 March 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 During protein synthesis, mRNA and tRNA undergo coupled translocation through the ribosome in a process that is catalyzed by elongation factor G (EF-G). On the basis of cryo-EM reconstructions, counterclockwise and clockwise rotational movements between the large and small ribosomal subunits have been implicated in a proposed ratcheting mechanism to drive the unidirectional movement of translocation. We used a combination of two fluorescence-based approaches to study the timing of these events, intersubunit fluorescence resonance energy transfer measurements to observe relative rotational movement of the subunits, and a fluorescence quenching assay to monitor translocation of mRNA. Binding of EF-G–GTP first induces rapid counterclockwise intersubunit rotation, followed by a slower, clockwise reversal of the rotational movement. We compared the rates of these movements and found that mRNA translocation occurs during the second, clockwise rotation event, corresponding to the! transition from the hybrid state to the classical state. View full text Figures at a glance * Figure 1: Hybrid-state model of translocation and experimental design. () Schematic of intersubunit rotation and tRNA movement in pretranslocation ribosomes. Movement of mRNA is not depicted here. Spontaneous, nonproductive forward and reverse intersubunit rotation, coupled to fluctuations between the classical and hybrid states of tRNA binding, can occur without EF-G. () In the presence of EF-G, intersubunit rotation is coupled to translocation of tRNA on the 30S subunit. In the first step of translocation, EF-G can either induce hybrid state formation in nonrotated, classical-state ribosomes or bind directly to rotated, hybrid-state ribosomes. In the second step, tRNAs are translocated on the 30S subunit. () Experimental design. Intersubunit rotation was measured by changes in the FRET signal between a donor dye (blue) attached to protein S6 on the 30S subunit and an acceptor dye (orange) attached to protein L9 on the 50S subunit. Counterclockwise rotation, accompanying formation of the hybrid-state intermediate, leads to a decrease in FRET; ! reverse, clockwise movement increases the efficiency of energy transfer. mRNA translocation was measured by quenching of the fluorescence of a pyrene dye (yellow) attached to position +9 of the mRNA as it enters the ribosome. * Figure 2: Kinetics of intersubunit rotation and mRNA translocation. (–) Kinetics of intersubunit rotation followed by FRET (acceptor fluorescence; ,). Kinetics of mRNA translocation measured by quenching of fluorescence of pyrene attached to the mRNA24 (). Pretranslocation ribosomes containing deacylated elongator tRNAMet in the P site and N-Ac-Phe-tRNAPhe in the A site were rapidly mixed with EF-G–GTP (,). Intersubunit FRET signal (acceptor fluorescence) for S6-D/L9-A (red) and S11-D/L9-A (green) ribosomes (). Quenching of fluorescence of pyrene-labeled mRNA in wild-type (WT, blue) and S6-D/L9-A (red) ribosomes (). Pretranslocation ribosomes containing initiator tRNAfMet in the P site and N-Ac-Phe-tRNAPhe in the A site were rapidly mixed with EF-G–GTP (,). Intersubunit FRET signal (acceptor fluorescence) for S6-D/L9-A ribosomes (red, ). Quenching of fluorescence of pyrene-labeled mRNA in wild-type (blue) and S6-D/L9-A (red) ribosomes (). Single-exponential fits for FRET traces and double-exponential fits for mRNA quenching are black l! ines. * Figure 3: Resolution of the first and second steps of intersubunit rotation with antibiotics. (–) Kinetics of intersubunit rotation (,) and mRNA translocation (,) with spectinomycin (,) and hygromycin B (,). Pretranslocation ribosomes containing deacylated initiator tRNAfMet in the P site and N-Ac-Phe-tRNAPhe in the A site were preincubated with either spectinomycin (,) or hygromycin B (,) and rapidly mixed with EF-G–GTP. The FRET signal (acceptor fluorescence) for S6-D/L9-A ribosomes is red; quenching of pyrene-labeled mRNA in wild-type (WT) and S6-D/L9-A ribosomes is blue and green, respectively. Single-exponential (intersubunit FRET) and double-exponential (mRNA translocation) fits are black lines. * Figure 4: Stabilization of the rotated, hybrid-state conformation by binding of EF-G–GDP in the presence of fusidic acid. Pretranslocation ribosomes containing initiator tRNAfMet in the P site and N-Ac-Phe-tRNAPhe in the A site were rapidly mixed with EF-G–GDP and fusidic acid (Fus). The intersubunit FRET signal (acceptor fluorescence) for S6-D/L9-A ribosomes is red. Quenching of pyrene-labeled mRNA fluorescence in wild-type (WT) and S6-D/L9-A ribosomes is blue and green, respectively. * Figure 5: Effect of inhibition of EF-G release on the second (clockwise) step of intersubunit rotation and mRNA translocation. Pretranslocation ribosomes containing elongator tRNAMet in the P site and N-Ac-Phe-tRNAPhe in the A site were rapidly mixed with EF-G–GDPNP or EF-G–GTP and fusidic acid (Fus). () FRET signal (acceptor fluorescence) for S6-D/L9-A ribosomes. () Quenching of pyrene-labeled mRNA in wild-type ribosomes. EF-G–GDPNP traces are blue and those for EF-G–GTP and fusidic acid are red. Single-exponential (intersubunit FRET) and double-exponential (mRNA translocation) fits are black lines. Author information * Abstract * Author information * Supplementary information Affiliations * Center for Molecular Biology of RNA, University of California, Santa Cruz, Santa Cruz, California, USA. * Dmitri N Ermolenko & * Harry F Noller * Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, California, USA. * Dmitri N Ermolenko & * Harry F Noller * Present address: Department of Biochemistry and Biophysics and Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA. * Dmitri N Ermolenko Contributions D.N.E. and H.F.N. designed the research; D.N.E. carried out the experiments; D.N.E. and H.F.N. analyzed the data and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Harry F Noller Author Details * Dmitri N Ermolenko Search for this author in: * NPG journals * PubMed * Google Scholar * Harry F Noller Contact Harry F Noller Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (60K) Supplementary Figure 1 Additional data
• Structural basis for engagement by complement factor H of C3b on a self surface
  - Nat Struct Mol Biol 18(4):463-470 (2011)
  Nature Structural & Molecular Biology | Article Structural basis for engagement by complement factor H of C3b on a self surface * Hugh P Morgan1, 6 * Christoph Q Schmidt2, 6 * Mara Guariento2 * Bärbel S Blaum2, 5 * Dominic Gillespie1 * Andrew P Herbert2, 5 * David Kavanagh3 * Haydyn D T Mertens4 * Dmitri I Svergun4 * Conny M Johansson2 * Dušan Uhrín2 * Paul N Barlow2 * Jonathan P Hannan1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:463–470Year published:(2011)DOI:doi:10.1038/nsmb.2018Received06 October 2010Accepted14 January 2011Published 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 Complement factor H (FH) attenuates C3b molecules tethered by their thioester domains to self surfaces and thereby protects host tissues. Factor H is a cofactor for initial C3b proteolysis that ultimately yields a surface-attached fragment (C3d) corresponding to the thioester domain. We used NMR and X-ray crystallography to study the C3d–FH19–20 complex in atomic detail and identify glycosaminoglycan-binding residues in factor H module 20 of the C3d–FH19–20 complex. Mutagenesis justified the merging of the C3d–FH19–20 structure with an existing C3b–FH1–4 crystal structure. We concatenated the merged structure with the available FH6–8 crystal structure and new SAXS-derived FH1–4, FH8–15 and FH15–19 envelopes. The combined data are consistent with a bent-back factor H molecule that binds through its termini to two sites on one C3b molecule and simultaneously to adjacent polyanionic host-surface markers. View full text Figures at a glance * Figure 1: Introduction to C3b/C3d and complement factor H. () The central event in complement activation is the cleavage of C3 to C3b by C3 convertase, accompanied by attachment to surfaces mediated by the TED. In the presence of additional complement regulatory molecules, C3b may be further degraded sequentially to iC3b, C3c, C3dg and C3d. C3d corresponds to the TED and remains bound to the cell surface. () Complement factor H has 20 CCPs and multiple binding sites for different ligands. Disease-linked mutations have been reported throughout factor H. Several recombinant fragments of factor H were used in the current studies and are indicated by black brackets. FD, factor D; FI, factor I; DAF, decay-accelerating factor; CR1, complement receptor type 1; CUB, complement C1r-C1s; UEGF, urinary epidermal growth factor; BMP1, bone morphogenetic protein 1. * Figure 2: Structure of the C3d–FH19–20 complex showing interacting domains and overall molecular architecture. All interacting amino-acid residues are shown as sticks; hydrogen bonds are represented by dashed lines. The resolution of the electron density (2Fo − Fc map) map is 2.1 Å and it is contoured at 1σ. () Modules 19 (residues 1108–1166) and 20 (residues 1167–1231) of factor H are shown in green. Amino acids belonging to two interacting helices (α7 (cyan, residues 170–189) and α4 (purple, residues 106–118)) of C3d (gray) form the majority of interactions with factor H. () Electrostatic surface representations of C3d and FH19–20 (calculated using the APBS plug-in for PyMOLl51) rotated 90° counter-clockwise and clockwise, respectively, from the orientations shown in . (,) Enlarged views of the interface between C3d and FH19–20. * Figure 3: Heteronuclear NMR spectroscopy used to map the C3d and dp8 binding sites on FH19–20. () The percentage broadening for unequivocally assigned backbone amides upon addition of C3d. Red bars, >90% broadening; white bars, proline residues; gray bars, residues whose assignments were missing or whose intensities were weak in free FH19–20. () Amides that show line-broadening of >90% are shown as red spheres on the structure of FH19–20 (2G7I). The sizes of the spheres have been adjusted to correlate with the degree of line-broadening of each amide cross-peak. Larger spheres represent complete disappearance of the signal; smaller spheres represent signals that are broadened by >90% but are still detectable. () Combined amide chemical shift changes upon addition of 8.5-fold excess of dp8. Blue bars indicate shift changes larger than the threshold (double the average shift change; corresponding residues mapped onto the structure of FH19–20 in Fig. 5d), white and gray bars as in . * Figure 4: SPR studies of FH19–20 mutants binding to C3d. () Sensorgram showing the binding of wild-type FH19–20 to amine-coupled plasma-derived C3d. () KD fitting of . () Sensorgram showing the weak binding of FH19–20 D1119G to amine-coupled plasma-derived C3d. () Summary of affinity constants of factor H fragments and mutants for recombinant C3d. () Surface representation of FH19–20 (2G7I) highlighting residues corresponding to mutations that change C3b binding31. () Surface representation of FH19–20 (2G7I) highlighting residues corresponding to mutations that change C3d binding. Ser1191 and Val1197 are buried residues. Val1197 has been analyzed in the context of the double mutant S1191L V1197A. Leu1189 has been analyzed in the context of two mutants: L1189R and L1189F. () Summary of the KD values for complexes of mutants of FH19–20 and C3d mutants E117A (E1110A), D122A (D1115A) and E160A (E1153A), I164A (I1157A, with wild-type FH19–20 only) and E117A D122A (E1110A D1115A, with wild-type FH19–20 only). The KD of the! latter was ~30 mM. *Corresponding KD value was extrapolated. Error bars for KD measurements indicate s.e.m. * Figure 5: Potential model of factor H engagement with surface-bound C3b. () Superposition (C3d on TED) of the C3d–FH19–20 complex and C3b–FH1–4 complex showing the close proximity of factor H modules 4 and 19. () The FH19–20–C3d structure (red and cyan) superimposed (C3d on TED) onto the FH1–4–C3b structure (tan and gray). This factor H model has been constructed by juxtaposition or superposition of the structures of FH5 (yellow) and FH6–8 (yellow-orange) and the SAXS-shape envelopes of FH8–15 (bright orange) and FH15–19 (orange). Structures are shown in cartoon representation; SAXS shape envelopes are shown in mesh. The FH1–4 SAXS shape envelope (pale yellow) is superimposed onto the C3b–FH1–4 structure. () Close up view of the α4-α5 loop and the α6-α7 loop of C3d. Highlighted in orange are Pro121 (Pro1114), Asp122 (Asp1115), Cys165 (Cys1158) and Gln168 (Gln1161), for which disease-associated mutations have been reported. () Close-up view of the interaction of FH19–20 with TED, highlighting residues that are als! o likely to interact with self-surface markers. Amides that undergo considerable chemical shift perturbations upon exposure to dp8 are highlighted as blue spheres (corresponding to their nitrogen atoms). Residues Lys1188 and Arg1231, which show the greatest perturbations upon addition of dp8, are indicated. * Figure 6: The locations of missense mutations associated with the development of aHUS (and of Q1139A) mapped onto the structure of the C3d–FH19–20 complex. Blue sticks, side chains of C3b missense mutations; cyan sticks, side chains of FH19–20 missense mutations. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3OXU * 3OXU Referenced accessions Protein Data Bank * 2WII * 2G7I * 1C3D * 2WII * 2G7I * 1C3D Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Hugh P Morgan & * Christoph Q Schmidt Affiliations * Institute of Structural and Molecular Biology, School of Biological Sciences, King's Buildings, University of Edinburgh, Edinburgh, UK. * Hugh P Morgan, * Dominic Gillespie & * Jonathan P Hannan * Edinburgh Biomolecular NMR Unit, EaStCHEM, School of Chemistry, University of Edinburgh, Edinburgh, UK. * Christoph Q Schmidt, * Mara Guariento, * Bärbel S Blaum, * Andrew P Herbert, * Conny M Johansson, * Dušan Uhrín & * Paul N Barlow * The Institute of Human Genetics, University of Newcastle upon Tyne, Newcastle upon Tyne, UK. * David Kavanagh * European Molecular Biology Laboratory, Hamburg Outstation, Hamburg, Germany. * Haydyn D T Mertens & * Dmitri I Svergun * Present addresses: Interfaculty Institute for Biochemistry, University of Tübingen, Tübingen, Germany (B.S.B.); The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Liverpool, UK (A.P.H.). * Bärbel S Blaum & * Andrew P Herbert Contributions M.G., C.Q.S., D.K. and A.P.H. engineered and produced factor H proteins; J.P.H. and D.G. engineered and produced C3d proteins; C.M.J. generated dp8 heparin fragments. J.P.H. and D.G. crystallized the C3d–FH complex; H.P.M. and J.P.H. collected crystallographic data; H.P.M. determined and refined the structure; B.S.B. produced 15N-labeled factor H and carried out NMR studies of the FH–C3d and FH–dp8 interactions; B.S.B. and A.P.H. carried out the hemolysis assay; J.P.H. carried out the C3d-dp8 competition ELISA; C.Q.S. and M.G. carried out the SPR measurements; H.D.T.M. and D.I.S. carried out the SAXS analysis; D.U., J.P.H. and P.N.B. conceived and supervised the project; H.P.M., C.Q.S., M.G., B.S.B., D.U., J.P.H. and P.N.B. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Paul N Barlow or * Jonathan P Hannan Author Details * Hugh P Morgan Search for this author in: * NPG journals * PubMed * Google Scholar * Christoph Q Schmidt Search for this author in: * NPG journals * PubMed * Google Scholar * Mara Guariento Search for this author in: * NPG journals * PubMed * Google Scholar * Bärbel S Blaum Search for this author in: * NPG journals * PubMed * Google Scholar * Dominic Gillespie Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew P Herbert Search for this author in: * NPG journals * PubMed * Google Scholar * David Kavanagh Search for this author in: * NPG journals * PubMed * Google Scholar * Haydyn D T Mertens Search for this author in: * NPG journals * PubMed * Google Scholar * Dmitri I Svergun Search for this author in: * NPG journals * PubMed * Google Scholar * Conny M Johansson Search for this author in: * NPG journals * PubMed * Google Scholar * Dušan Uhrín Search for this author in: * NPG journals * PubMed * Google Scholar * Paul N Barlow Contact Paul N Barlow Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan P Hannan Contact Jonathan P Hannan 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, Supplementary Tables 1–3 and Supplementary Methods Additional data
• The structural basis for MCM2–7 helicase activation by GINS and Cdc45
  - Nat Struct Mol Biol 18(4):471-477 (2011)
  Nature Structural & Molecular Biology | Article The structural basis for MCM2–7 helicase activation by GINS and Cdc45 * Alessandro Costa1, 2 * Ivar Ilves1 * Nele Tamberg1 * Tatjana Petojevic1, 3 * Eva Nogales1, 2, 4, 5 * Michael R Botchan1, 2 * James M Berger1, 2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:471–477Year published:(2011)DOI:doi:10.1038/nsmb.2004Received22 October 2010Accepted09 December 2010Published online06 March 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 Two central steps for initiating eukaryotic DNA replication involve loading of the Mcm2–7 helicase onto double-stranded DNA and its activation by GINS–Cdc45. To better understand these events, we determined the structures of Mcm2–7 and the CMG complex by using single-particle electron microscopy. Mcm2–7 adopts two conformations—a lock-washer-shaped spiral state and a planar, gapped-ring form—in which Mcm2 and Mcm5 flank a breach in the helicase perimeter. GINS and Cdc45 bridge this gap, forming a topologically closed assembly with a large interior channel; nucleotide binding further seals off the discontinuity between Mcm2 and Mcm5, partitioning the channel into two smaller pores. Together, our data help explain how GINS and Cdc45 activate Mcm2–7, indicate that Mcm2–7 loading may be assisted by a natural predisposition of the hexamer to form open rings, and suggest a mechanism by which the CMG complex assists DNA strand separation. View full text Figures at a glance * Figure 1: Mcm2–7 exists in two states. (,) Reference-free class averages and corresponding forward projections of notched-ring or lock-washer reconstructions. () 3D reconstruction of the notched ring viewed from the AAA+ face (full view, left; slab view, right), the side and the N-terminal face. Mcm homology models are fitted into the reconstruction. () 3D structure of the lock-washer ring viewed from the AAA+ (full or slab view), side and N-terminal faces, and fitted with homology models. * Figure 2: Subunit mapping of Mcm2–7. () Projection per class average matching of the Mcm2–7 lock-washer state with an N-terminal MBP tag on Mcm6. Orange arrowheads mark additional density observed on the ring. () Class averages of Mcm2–7 containing MBP-tagged Mcm3. Orientations match those in . Blue arrowheads mark additional density observed on the ring. () Class-average projection matching of Mcm2–7 containing MBP-tagged Mcm4. The tag appears detached from the ring density (green arrowheads), consistent with the presence of a 154-residue N-terminal tail on Mcm4. () Possible subunit arrangements for gapped Mcm2–7 complexes as defined by MBP-tagged Mcm3 and Mcm6. The correct configuration further defined by MBP-tagged Mcm4 is shown in color. * Figure 3: CMG contains a notched, planar Mcm2–7 ring that is sealed upon nucleotide binding. () Class-average projection matching for apo CMG. () AAA+ view of apo CMG, showing a discontinuity between Mcm2–5. () Class-average projection matching for ADP·BeF3-bound CMG. () C-terminal AAA+ view of ADP·BeF3-bound CMG, showing a pinched-off gap between Mcm2–5. () Class averages of apo (top) or ADP·BeF3-bound CMG (bottom) in the same orientation, showing the presence (yellow arrowhead) or absence (gray arrowhead) of an Mcm2–5 gap. * Figure 4: Mcm-subunit mapping in the CMG. (–) Class-average projection matching for ADP·BeF3-bound CMG with (unboxed) or without (boxed) N-terminal MBP tags (arrowheads) on Mcm6 (), Mcm3 () or Mcm4 (). Mcm4 is distal from GINS and Cdc45 (see also Supplementary Fig. 5). (–) Unfiltered 3D reconstructions of ADP·BeF3-bound CMG with an N-terminal MBP tag on either Mcm6 () or Mcm3 () and compared to both untagged CMG () and a CMG complex with a 154-amino-acid deletion of the N-terminal Mcm4 tail (). Extra density attributed to MBP on Mcm6 and Mcm3 is colored orange or light blue, respectively; only a portion of the Mcm4 tail is observable in the full-length constructs and is highlighted in green. () Density corresponding to MBP on Mcm3 (light blue) or Mcm6 (orange), cut out from the CMG density maps and compared to the density map calculated from the MBP crystal structure, PDB entry 3HPI51, filtered to 30 Å and showed at 5σ (gray). * Figure 5: Structure docking into CMG reconstructions. (,) From left to right: N-terminal, side and C-terminal (AAA+ domain) views of apo () or ADP·BeF3-bound CMG (). The red double arrow in indicates the location of a discontinuity in the AAA+ tier of Mcm2–7 in apo CMG. GINS is colored white; unoccupied density assigned to Cdc45 is solid gray. * Figure 6: GINS contacts within the CMG. () Crystal structures of the four individual subunits of the GINS complex37. As Sld5 and Psf1 are close homologs, an orientation for the β-domain in Psf1 was generated using that seen in Sld5. The related β-domains of Psf1 and Psf3 are shown in black and highlighted with gold ovals. Psf2 and Psf3 share close structural similarity; a related helical element (α1) is shown in black and highlighted with gold ovals. () Three views of the crystal structure of GINS docked into the CMG density. () Zoomed-out (left) and close-up (right) views of GINS engaged with Cdc45 and the N-terminal domains of Mcm3 and Mcm5. () Zoomed-out (left) and close-up (right) views of GINS and its interactions with Cdc45 and the AAA+ domains of Mcm3 and Mcm5. () Nucleotide-triggered movements in the GINS-AAA+ domain interaction region appear to seal off the Mcm2–7 central channel. * Figure 7: CMG interactions. () Expression levels of individual proteins used for testing CMG formation. () Immunoprecipitation (IP) experiments testing CMG stability. 1, wild-type CMG; 2, CMG with a C-terminal truncation in Psf1; 3, CMG without MCM5; 4, CMG without Cdc45. Mcm3 immunoprecipitation yields the intact CMG complex, whereas deletion of the Psf1 β domain (Psf1ΔC, amino acids 1–139), or withholding Mcm5 or Cdc45, disrupts CMG formation. () Expression levels of individual proteins used for testing GINS formation. () IP experiments testing GINS stability. The C-terminal β-domain of Psf1 is dispensable for GINS formation. () Summary of interactions in the CMG. Subunits are demarcated by spheres, which have been placed into the 3D reconstruction obtained for the ADP·BeF3-bound complex (transparent surface). The N- and C-terminal regions of each Mcm subunit are highlighted separately and labeled 2N–7N and 2C–7C, respectively; other subunits are labeled by their full name. Dotted lines sho! w noncovalent interactions observed in the complex and are colored as follows: black, intra-MCM; blue, intra-GINS; green, GINS with Cdc45; red, GINS and Cdc45 with Mcm2–7. * Figure 8: Model for Mcm2–7 activation and function. () Free Mcm2–7 can exist in either an open, lock-washer or notched, planar configuration. Each form shows a discontinuity between Mcm2 and Mcm5. Binding of GINS–Cdc45 stabilizes the notched, planar Mcm2–7 state, whereas ATP binding promotes ring closure. () ORC, Cdc6 and Cdt1 load a preopened Mcm2–7 assembly onto dsDNA as an inactive double hexamer. GINS and Cdc45 bind to Mcm2–7, concomitant with an isomerization that creates or stabilizes melted DNA. The side channel formed by the GINS–Cdc45 subcomplex likely prevents DNA escape from Mcm2–7 and may help partition the lagging DNA strand from its complement following unwinding. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA. * Alessandro Costa, * Ivar Ilves, * Nele Tamberg, * Tatjana Petojevic, * Eva Nogales, * Michael R Botchan & * James M Berger * California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, California, USA. * Alessandro Costa, * Eva Nogales, * Michael R Botchan & * James M Berger * Department of Biology, Chemistry and Pharmacy, Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany. * Tatjana Petojevic * Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA. * Eva Nogales * Life Science Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. * Eva Nogales Contributions A.C., I.I., M.R.B. and J.M.B. conceived the general ideas for this study. All authors planned experiments. A.C. did all electron microscopy single-particle reconstruction and molecular modeling supervised by J.M.B. and E.N. I.I., N.T. and T.P. did cloning, baculovirus construction and protein purification supervised by M.R.B. A.C., M.R.B. and J.M.B. wrote the manuscript. All authors provided editorial input. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * James M Berger or * Michael R Botchan Author Details * Alessandro Costa Search for this author in: * NPG journals * PubMed * Google Scholar * Ivar Ilves Search for this author in: * NPG journals * PubMed * Google Scholar * Nele Tamberg Search for this author in: * NPG journals * PubMed * Google Scholar * Tatjana Petojevic Search for this author in: * NPG journals * PubMed * Google Scholar * Eva Nogales Search for this author in: * NPG journals * PubMed * Google Scholar * Michael R Botchan Contact Michael R Botchan Search for this author in: * NPG journals * PubMed * Google Scholar * James M Berger Contact James M Berger Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (5M) The Mcm2-7 complex morphing between a planar-notched and a spiral-lockwasher configuration. * Supplementary Movie 2 (4M) The CMG complex morphing between the apo and nucleotide-bound state PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–8 Additional data
• Rudimentary G-quadruplex–based telomere capping in Saccharomyces cerevisiae
  - Nat Struct Mol Biol 18(4):478-485 (2011)
  Nature Structural & Molecular Biology | Article Rudimentary G-quadruplex–based telomere capping in Saccharomyces cerevisiae * Jasmine S Smith1, 2 * Qijun Chen1 * Liliya A Yatsunyk3 * John M Nicoludis3 * Mark S Garcia1, 2 * Ramon Kranaster4 * Shankar Balasubramanian4, 5, 6 * David Monchaud7 * Marie-Paule Teulade-Fichou7 * Lara Abramowitz1, 2 * David C Schultz8 * F Brad Johnson1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:478–485Year published:(2011)DOI:doi:10.1038/nsmb.2033Received02 April 2010Accepted29 December 2010Published online13 March 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 Telomere capping conceals chromosome ends from exonucleases and checkpoints, but the full range of capping mechanisms is not well defined. Telomeres have the potential to form G-quadruplex (G4) DNA, although evidence for telomere G4 DNA function in vivo is limited. In budding yeast, capping requires the Cdc13 protein and is lost at nonpermissive temperatures in cdc13-1 mutants. Here, we use several independent G4 DNA–stabilizing treatments to suppress cdc13-1 capping defects. These include overexpression of three different G4 DNA binding proteins, loss of the G4 DNA unwinding helicase Sgs1, or treatment with small molecule G4 DNA ligands. In vitro, we show that protein-bound G4 DNA at a 3′ overhang inhibits 5′→3′ resection of a paired strand by exonuclease I. These findings demonstrate that, at least in the absence of full natural capping, G4 DNA can play a positive role at telomeres in vivo. View full text Figures at a glance * Figure 1: Overexpression of the G4 DNA binding protein Stm1 rescues growth defects caused by telomere uncapping and is independent of RAD52-dependent homologous recombination. () Growth of cdc13-1 mutants carrying pSTM1 or vector alone at permissive temperature (PT, 22 °C) or at semipermissive temperature (SPT, 30 °C). () pSTM1 overexpression rescues impaired growth caused by telomere uncapping in stn1-154 mutants at SPT. () Growth of cdc13-1 rad52Δ mutants carrying pSTM1 or vector. For each assay, serial dilutions of cells were spotted on selective medium and grown for 3 d (,) or 4 d (). () Top: map of a typical telomere containing two tandem subtelomeric Y′ elements, separated by interstitial telomere repeats. Bottom: telomere Southern blots of samples grown in liquid culture at SPT for 2 d. Type I and type II survivors of telomerase inactivation are shown for comparison. The different sizes of internal Y′ fragments are due to short and long forms of Y′. Lanes 1–5 and 6–9 are sections from the same Southern blot. * Figure 2: Expression of two additional, distinct G4 DNA binding proteins rescues the cdc13-1 temperature-sensitive phenotype. () The G4 DNA–binding Sgs1 RQC domain, overexpressed from a plasmid and under the control of the GAL1 promoter (pRQC), or vector control were transformed into the indicated strain backgrounds and tested in spot assays. () A 5× HA-tagged Sgs1 RQC domain is enriched at telomeres of cdc13-1 mutants. The primer sets used were directly adjacent to the telomeres (subtelomeric Y′ repeat) or a control, centromere-proximal primer set (to a portion of SWC4, which has no QFP). The telomeric/centromeric ratio is listed above bars. Error bars are ± 1 s.d., and essentially the same result was obtained in three independent ChIP experiments. Mock IP indicates that cdc13-1 sgs1Δ cells with the RQC-expressing vector were ChIPed without antibody. () GAL-induced overexpression of the G4 DNA binding single-chain antibody (scFv), HF1 or empty vector in cdc13-1 mutants. For each assay, serial dilutions of cells were spotted on selective medium containing 2% galactose and grown at the indica! ted temperatures for 4 d. () The 13xMyc-tagged HF1 scFv is enriched at telomeres of cdc13-1 mutants. DNA was amplified as described in (). Error bars are ± 1 s.d., and essentially the same result was obtained in three independent ChIP experiments. * Figure 3: Loss of the SGS1 activities associated with G4 DNA binding and unwinding rescues cdc13-1 temperature sensitivity and is independent of RAD52-dependent homologous recombination. () Growth of cdc13-1 rad24Δ rad52Δ, either SGS1 or sgs1Δ, at permissive temperature versus SPT. () Map of specific Sgs1 domains mutated or deleted in this study. () Deletion of the G-quadruplex binding RQC domain alone is sufficient to rescue the cdc13-1 growth defect. () Loss of Sgs1 helicase activity (K706A point mutation, denoted sgs1-hd) is sufficient to rescue cdc13-1 temperature sensitivity. () Sgs1 is the sole component of the Sgs1–Top3–Rmi1 complex that, when lost, is sufficient to rescue the cdc13-1 temperature sensitive phenotype. For each assay, serial dilutions of cells were spotted on YPAD (,) or selective medium (,) and grown at the indicated temperatures for 3 d (,,) or 4 d (). * Figure 4: Telomere-proximal single-stranded (ss) DNA accumulation at NPT (37 °C) is attenuated by two G4 DNA-stabilizing mechanisms. (,) ssDNA measurements in the context of STM1 overexpression () or sgs1 deletion () in cdc13-1 rad24Δ rad52Δ backgrounds. Subtelomeric G-strand ssDNA was probed with a complementary end-labeled ssDNA probe against the Y′ element and quantified by normalizing hybridization signals of native to denatured samples. Each sample was spotted in triplicate, and standard errors are shown. Each graph is one representative experiment. The apparent difference in the timing of ssDNA accumulation between the experiments reflects the time points examined rather than true experimental variability. (,) Attenuation of Rad53 phosphorylation by Stm1 overexpression or sgs1 deletion in cdc13-1 rad24Δ rad52Δ cells incubated at NPT (37 °C). Treatment of wild-type cells with 0.033% methyl methanesulfonate (MMS) provides a positive control for Rad53 phosphorylation (, lane 1). * Figure 5: Diminished rescue of cdc13-1 growth at SPT by sgs1 deletion in cells with mutant telomerase RNA templates that decrease QFP at telomeres. () cdc13-1 rad24Δ rad52Δ tlc1Δ cells, either SGS1 or sgs1Δ, and with plasmid-borne wild-type TLC1 or the uCu or CuA mutant tlc1 alleles, were spotted on SC-HIS medium and grown for 4 d. The sequence of the TLC1 template, including the mutated region (bold), is as follows: 3′-CACACACCACCAC-5′. () CD spectra of representative tlc1 mutant telomere sequences and their corrected counterparts (see Table 1). () Thermal difference spectra (TDS) of mutant versus corrected sequences, shown as the difference between molar extinction coefficients. G4 DNA yields a negative peak at 295 nm and positive peaks at 273 and 242 nm. All four corrected sequences showed evidence of G4 DNA formation by means of CD (parallel quadruplex for 1-corr and mixed parallel and antiparallel for 2, 3 and 4-corr) and TDS. () Thermal denaturation of oligonucleotides was followed by CD absorbance at 263 nm (for oligonucleotides 1, 1-corrected (corr), 2 and 2-corr) or 290 nm (for oligonucleotides 3, 3-cor! r, 4 and 4-corr). * Figure 6: G4 DNA-binding proteins and G4 DNA-forming sequences cooperate to inhibit end-resection by Exo1 in vitro. () Synthetic substrates having 3′ overhangs that form (bottom) or do not form (top) G-quadruplex DNA. Each contains 51 bp of identical duplex DNA. The 5′ end of each top strand was biotinylated (B) to inhibit resection from the incorrect end, and the terminal base of each bottom strand was labeled with α-32P-dCTP. We illustrate a parallel intramolecular quadruplex (bottom), although this may not be the actual or only structure formed by this overhang. () Analysis of overhang substrates by CD shows a mixture of parallel and anti-parallel G4 DNA (GGG overhang) and a possible GA homoduplex (GAG overhang) (see Supplementary Fig. 6) under the exact conditions used for the Exo1 assay. () Thermal difference spectra of GGG and GAG yield a G-quadruplex-specific signal for only the GGG overhang. (,) Both Stm1 () and HF1 () cooperate with G4 DNA overhang to partially rescue Exo1 5′→3′ resection. Quantification of the fraction of remaining full-length sequence; shown are the ! averages of two () or three () independent experiments with standard errors and P-values from unpaired two-tailed Student's t-tests. () Quantification of protection by T4 gene 32 protein (T4g32p). An average of two experiments is shown. P > 0.7 for all concentrations of T4g32p. * Figure 7: Model of telomere capping in which G4 DNA cooperates with G-quadruplex binding proteins (G4BP) to inhibit exonucleolytic degradation and checkpoint activation caused by loss of Cdc13 function. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. * Jasmine S Smith, * Qijun Chen, * Mark S Garcia, * Lara Abramowitz & * F Brad Johnson * Cell and Molecular Biology Group, Biomedical Graduate Studies, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. * Jasmine S Smith, * Mark S Garcia, * Lara Abramowitz & * F Brad Johnson * Department of Chemistry and Biochemistry, Swarthmore College, Swarthmore, Pennsylvania, USA. * Liliya A Yatsunyk & * John M Nicoludis * Department of Chemistry, University of Cambridge, Cambridge, UK. * Ramon Kranaster & * Shankar Balasubramanian * School of Clinical Medicine, University of Cambridge, Cambridge, UK. * Shankar Balasubramanian * Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge, UK. * Shankar Balasubramanian * Institut Curie, Section de Recherche, Centre National de la Recherche Scientifique, UMR176, Centre Universitaire Paris XI, Orsay, France. * David Monchaud & * Marie-Paule Teulade-Fichou * Protein Expression, Libraries and Molecular Screening Facility, The Wistar Institute, Philadelphia Pennsylvania, USA. * David C Schultz Contributions J.S.S. and F.B.J. conceived of and carried out experiments, interpreted results and wrote the manuscript. L.A.Y. and J.M.N. conducted and designed CD, thermal difference spectroscopy and fluorescence resonance energy transfer experiments, along with F.B.J. and J.S.S., and provided comments on the manuscript. R.K. and S.B. provided the HF1 cDNA and protein, designed and conducted CD and ELISA experiments (Supplementary Fig. 3) and provided comments on the manuscript. D.C.S. provided purified proteins and comments on the manuscript. D.M. and M.-P. T.-F. synthesized the bisquinolinium G4 DNA ligands and provided comments on the manuscript. Q.C., M.S.G. and L.A. carried out experiments. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * F Brad Johnson Author Details * Jasmine S Smith Search for this author in: * NPG journals * PubMed * Google Scholar * Qijun Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Liliya A Yatsunyk Search for this author in: * NPG journals * PubMed * Google Scholar * John M Nicoludis Search for this author in: * NPG journals * PubMed * Google Scholar * Mark S Garcia Search for this author in: * NPG journals * PubMed * Google Scholar * Ramon Kranaster Search for this author in: * NPG journals * PubMed * Google Scholar * Shankar Balasubramanian Search for this author in: * NPG journals * PubMed * Google Scholar * David Monchaud Search for this author in: * NPG journals * PubMed * Google Scholar * Marie-Paule Teulade-Fichou Search for this author in: * NPG journals * PubMed * Google Scholar * Lara Abramowitz Search for this author in: * NPG journals * PubMed * Google Scholar * David C Schultz Search for this author in: * NPG journals * PubMed * Google Scholar * F Brad Johnson Contact F Brad Johnson Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Methods Additional data
• Dominant prion mutants induce curing through pathways that promote chaperone-mediated disaggregation
  - Nat Struct Mol Biol 18(4):486-492 (2011)
  Nature Structural & Molecular Biology | Article Dominant prion mutants induce curing through pathways that promote chaperone-mediated disaggregation * Susanne DiSalvo1 * Aaron Derdowski1 * John A Pezza1 * Tricia R Serio1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:486–492Year published:(2011)DOI:doi:10.1038/nsmb.2031Received16 August 2010Accepted16 December 2010Published online20 March 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 Protein misfolding underlies many neurodegenerative diseases, including the transmissible spongiform encephalopathies (prion diseases). Although cells typically recognize and process misfolded proteins, prion proteins evade protective measures by forming stable, self-replicating aggregates. However, coexpression of dominant-negative prion mutants can overcome aggregate accumulation and disease progression through currently unknown pathways. Here we determine the mechanisms by which two mutants of the Saccharomyces cerevisiaeSup35 protein cure the [PSI+] prion. We show that both mutants incorporate into wild-type aggregates and alter their physical properties in different ways, diminishing either their assembly rate or their thermodynamic stability. Whereas wild-type aggregates are recalcitrant to cellular intervention, mixed aggregates are disassembled by the molecular chaperone Hsp104. Thus, rather than simply blocking misfolding, dominant-negative prion mutants target mult! iple events in aggregate biogenesis to enhance their susceptibility to endogenous quality-control pathways. View full text Figures at a glance * Figure 1: PNM mutants are distinguished by their effective inhibitory ratios. () Wild-type (HSP104/HSP104) or heterozygous-disruption (HSP104/Δ) diploid strains expressing wild-type (WT) and PNM mutants (Q24R, G58D) from P35 at the indicated ratios were spotted on rich (1/4 YPD) or adenine-deficient (−ADE) media to analyze the [PSI+] phenotype. Wild-type [PSI+] and [psi−] diploids (74-D694) were included as controls. () To determine the frequency of prion loss, wild-type meiotic progeny (n ≥ 19 for each strain) were isolated from the diploids described in , and the percentage of [psi−] colonies was determined. * Figure 2: PNM mutants incorporate into wild-type aggregates and alter multiple events in prion propagation. () HA-tagged Sup35 (WT or mutants) expressed from P35 in haploid [PSI+] (+) or [psi−] (−) yeast strains, which also expressed untagged Sup35, was immunoprecipitated with anti-HA serum (Ab) and analyzed by SDS-PAGE and anti-Sup35 immunoblotting. () [psi−] haploids expressing Sup35 (WT or mutants) from P35 and a fluorescent reporter of translation termination efficiency (GST-UGA-DsRed-NLS) were mated to wild-type [PSI+] or [psi−] (74-D694) cells, and the percentage of fluorescent zygotes was scored. Error bars represent s.d. from three independent experiments, each analyzing at least 15 zygotes per cross (*P = 0.039 in comparison with WT). () The fluorescence intensities of zygotes isolated from the indicated crosses as described in were determined. Horizontal lines on boxes indicate 25th, 50th and 75th percentiles; error bars indicate 10th and 90th percentiles; dots represent outliers (n ≥ 30; *P = 0.0009). () Lysates from wild-type haploid strains expressing an add! itional copy of Sup35 (WT or mutants) from PtetO2 were incubated in SDS at the indicated temperatures before SDS-PAGE and quantitative immunoblotting for Sup35 (percentage of Sup35 at the indicated temperatures relative to 100 °C). Error bars represent s.d. (n ≥ 6, *P = 0.0003, **P = 0.0001, ***P = 0.008 in comparison with WT at the same temperature). * Figure 3: PNM mutants alter the accumulation of propagons but not their transmission. () Lysates from haploid wild-type yeast strains expressing Sup35 (WT or mutants) from PtetO2 were analyzed by SDD-AGE and immunoblotting for Sup35. Wild-type [PSI+]Strong, [PSI+]Weak and [psi−] yeast strains are shown as controls. () The number of propagons present in individual cells was determined for the indicated strains, as described in . Horizontal lines on boxes indicate 25th, 50th and 75th percentiles; error bars indicate 10th and 90th percentiles; dots represent outliers (n ≥ 39; *P ≤ 0.0001 in comparison with WT). () The proportion of Sup35 transmitted to daughter cells (circles) or to mother cells (squares) was determined by fluorescence loss in photobleaching (FLIP) of a [PSI+] strain expressing Sup35-GFP alone ([PSI+]) or with a second copy of Sup35 (WT-GFP + WT) or G58D (WT-GFP + G58D) from PtetO2. Error bars represent s.e.m. from three independent experiments, each analyzing at least 10 cells. * Figure 4: PNM expression promotes Hsp104-mediated disassembly of aggregates. () The number of propagons present in individual cells was determined for diploid [PSI+] strains expressing one endogenous copy of SUP35 and one copy of SUP35 (WT or mutants) from PtetO2 in a wild-type background (HSP104/HSP104) or in a heterozygous-disruption background (HSP104/Δ). Box plots are as described in the legend to Figure 3b. n ≥ 10 cells per strain; *P < 0.05, in comparison with the corresponding HSP104/HSP104 strain. () Lysates of diploid strains expressing Sup35 (WT) and PNM mutants in the indicated ratios were analyzed by SDD-AGE and immunoblotting for Sup35. Wild type (+) and heterozygous disruption of HSP104 (Δ) are indicated. () Lysates from BSC783/4c and 74-D694 [PSI+] haploids were analyzed by SDS-PAGE and quantitative immunoblotting for Hsp104 and Sup35. Error bars represent s.d. (n = 5, *P = 0.0031). () Haploid [PSI+] cells co-expressing endogenous SUP35 and a second copy of SUP35 (WT or mutants) from PtetO2 were transformed with pHSE-Hsp104 (cenP10! 4), SB590 (2μP104), pLH102 (2μPGPD) or a vector control, and the percentage of [psi−] colonies was scored after plasmid loss. Error bars represent s.d. from three independent experiments, each analyzing a total of 50 colonies (*P < 0.05, in comparison with vector control for the same strain). () Lysates from diploid [PSI+] strains expressing two copies of SUP35 (WT) or one wild-type and one mutant copy of SUP35 from PtetO2 were incubated in SDS at 50 °C or 100 °C before SDS-PAGE and quantitative immunoblotting for Sup35, and the ratio of signal before and after cycloheximide (CHX) treatment was determined. Error bars represent s.d. (n ≥ 3, *P = 0.029, **P = 0.011). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island, USA. * Susanne DiSalvo, * Aaron Derdowski, * John A Pezza & * Tricia R Serio Contributions S.D. and T.R.S. designed the experiments, analyzed the data and wrote the manuscript. S.D., A.D. and J.A.P. performed the experiments. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Tricia R Serio Author Details * Susanne DiSalvo Search for this author in: * NPG journals * PubMed * Google Scholar * Aaron Derdowski Search for this author in: * NPG journals * PubMed * Google Scholar * John A Pezza Search for this author in: * NPG journals * PubMed * Google Scholar * Tricia R Serio Contact Tricia R Serio Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (6M) Supplementary Figures 1–6 and Supplementary Tables 1–3 Additional data
• Strain conformation, primary structure and the propagation of the yeast prion [PSI+]
  - Nat Struct Mol Biol 18(4):493-499 (2011)
  Nature Structural & Molecular Biology | Article Strain conformation, primary structure and the propagation of the yeast prion [PSI+] * Katherine J Verges1, 2, 5 * Melanie H Smith1, 2, 3, 5 * Brandon H Toyama1, 2, 4 * Jonathan S Weissman1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:493–499Year published:(2011)DOI:doi:10.1038/nsmb.2030Received17 August 2009Accepted03 February 2011Published online20 March 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 Prion proteins can adopt multiple infectious strain conformations. Here we investigate how the sequence of a prion protein affects its capacity to propagate specific conformations by exploiting our ability to create two distinct infectious conformations of the yeast [PSI+] prion protein Sup35, termed Sc4 and Sc37. PNM2, a G58D point mutant of Sup35 that was originally identified for its dominant interference with prion propagation, leads to rapid, recessive loss of Sc4 but does not interfere with propagation of Sc37. PNM2 destabilizes the amyloid core of Sc37 and causes compensatory effects that slow prion growth but aid prion division and result in robust propagation of Sc37. By contrast, PNM2 does not affect the structure or chaperone-mediated division of Sc4 but interferes with its delivery to daughter cells. Thus, effective delivery of infectious particles during cell division is a crucial and conformation-dependent step in prion inheritance. View full text Figures at a glance * Figure 1: Characterization of in vivo prion phenotypes. () Expression levels of wild-type (WT) or PNM2 expressed from a plasmid in a [PSI+]Sc4 background. Expression was quantified by immunoblotting with a polyclonal antibody to SupNM. Values represent the mean ± s.d. for three experiments. () Representative in vivo prion phenotypes of yeast spotted on low adenine media. Wild-type Sup35 was replaced with (left) or was co-expressed with (middle) either WT or PNM2. () Enlarged view of the edge of the yeast spot. The presence of red sectors in [PSI+]Sc4 PNM2 indicates a loss of [PSI+]. () Quantification of loss of [PSI+] as determined by counting the number of [psi−] colonies after growing for 24 h in yeast extract peptone dextrose (YEPD) liquid medium. Values represent the mean ± s.d. for three experiments. () Representative in vivo prion phenotypes of yeast spotted on low-adenine medium. Wild-type Sup35 was replaced with PNM2, which was subsequently replaced with the WT through plasmid exchanges. * Figure 2: Schematic of the prion propagation cycle. (1) Fiber growth; (2) chaperone-mediated division; (3) partitioning to daughter cells. * Figure 3: Characterization of the physical properties of fibers formed in vitro. () A representative experiment monitoring the relative growth rates of wild-type (WT) and PNM2 SupNM. Polymerization of SupNM was conducted with 5% (w/w) WT seed of the specified conformation, and the rate of addition of SupNM monomers was monitored by Thioflavin T fluorescence. Data were normalized to initial and final intensities. () Growth rates of Sc4 and Sc37 PNM2 SupNM normalized to those of WT SupNM polymerized on the relevant seed. Initial time points were fitted to a line and the slope (initial growth rate) was calculated. Values represent mean ± s.d. for three experiments. (,) Thermal stability of WT and PNM2 fibers in Sc4 and Sc37 conformations. WT and PNM2 SupNM fibers in the Sc4 or Sc37 conformations were incubated at increasing temperatures, and samples were subjected to SDS-PAGE. Band intensities (susceptibility of aggregates to thermal solubilization) were plotted against temperature and fitted to a sigmoidal function. () Relative seeding efficacy of WT and ! PNM2 fibers in the Sc4 or Sc37 conformation before and after stirring for 30 or 60 min. Seeding efficacy was determined by monitoring the initial fiber growth rates of polymerization reactions using stirred samples as seeds. Values represent mean ± s.e.m. for three experiments. * Figure 4: H/D exchange of wild-type (WT) and PNM2 SupNM fibers. (–) Intensities for assigned and unambiguous peaks corresponding to residues 1–141 were plotted as the fraction of the non-exchanged intensity after 2 min (,) and 1 day (,) of exchange for WT (blue) and PNM2 (red) fibers in the Sc37 (,) and Sc4 (,) conformations. Unassigned and ambiguous residues are not shown. Gray line, estimated minimum peak intensity after complete exchange. * Figure 5: PNM2 fibers in the Sc4 conformation interact with the in vivo chaperone machinery. () HSP104 (top) and hsp104ΔNTD (bottom) were expressed in [PSI+]Sc4 yeast expressing wild-type (WT; left) or PNM2 (right) Sup35 and spotted onto low-adenine medium. () HSP104 and hsp104ΔNTD were expressed at increased levels with endogenous Hsp104 in [PSI+]Sc4 yeast expressing WT or PNM2 Sup35. 'Vector only' denotes transformation with an empty CEN/ARS plasmid. () SDD-AGE analysis of prion particle size in duplicate from lysates of [PSI+]Sc4 (lanes 1,2 and 5,6), [PSI+]Sc37 (lanes 3,4) and PNM2 [PSI+]Sc4 (lanes 7,8). SUP35 is genomic in lanes 1–4 and on a plasmid in lanes 5–8. Yeast express WT HSP104 in all lanes. () Schematic of the in vivo HAP-ClpPtrap reaction. () Representative blot of ClpPtrap affinity purification. WT Sup35 and ClpPtrap were expressed in backgrounds that were [PSI+]Sc4 or [psi−] expressing HAP or Hsp104. () Monitoring the translocation of PNM2. HAP and ClpPtrap were expressed in [PSI+]Sc4 cells that also expressed WT or PNM2 Sup35. Intensities o! f Sup35 elution signals from western blot (left) were normalized for input signal (right). () Monitoring translocation of PNM2 when co-expressed with WT. As in , but untagged WT Sup35 also expressed in cells expressing HA-tagged WT or PNM2 Sup35. The higher-molecular-weight band corresponds to HA-tagged Sup35. Intensities of tagged-Sup35 elution signals were normalized for input signal and for untagged-Sup35 elution signal. For Sup35 intensity, values are mean ± s.e.m. for three experiments. * Figure 6: PNM2 in the Sc4 conformation shows a defect in partitioning. (,) The number of propagons in the daughters was plotted against the number of propagons in the mothers for WT () and PNM2 () [PSI+]Sc4 backgrounds. Red dots represent mother-daughter pairs in which the mother contained propagons but the daughter did not. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Katherine J Verges & * Melanie H Smith Affiliations * Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, USA. * Katherine J Verges, * Melanie H Smith, * Brandon H Toyama & * Jonathan S Weissman * Howard Hughes Medical Institute, University of California, San Francisco, California, USA. * Katherine J Verges, * Melanie H Smith, * Brandon H Toyama & * Jonathan S Weissman * Graduate Group in Biophysics, University of California, San Francisco, California, USA. * Melanie H Smith * Present address: The Salk Institute for Biological Studies, La Jolla, California, USA. * Brandon H Toyama Contributions K.J.V., M.H.S. and J.S.W. designed this study and wrote the manuscript. J.S.W. supervised this work. K.J.V. and M.H.S. conducted the majority of the experiments. B.H.T. designed the H/X NMR experiments and acquired and analyzed the NMR data. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jonathan S Weissman Author Details * Katherine J Verges Search for this author in: * NPG journals * PubMed * Google Scholar * Melanie H Smith Search for this author in: * NPG journals * PubMed * Google Scholar * Brandon H Toyama Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan S Weissman Contact Jonathan S Weissman 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 and 2, Supplementary Tables 1 and 2, and Supplementary Methods Additional data
• Homology-directed Fanconi anemia pathway cross-link repair is dependent on DNA replication
  - Nat Struct Mol Biol 18(4):500-503 (2011)
  Nature Structural & Molecular Biology | Brief Communication Homology-directed Fanconi anemia pathway cross-link repair is dependent on DNA replication * Koji Nakanishi1 * Francesca Cavallo2 * Loïc Perrouault3, 4, 5 * Carine Giovannangeli3, 4, 5 * Mary Ellen Moynahan6 * Marco Barchi2 * Erika Brunet1, 3, 4, 5 * Maria Jasin1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:500–503Year published:(2011)DOI:doi:10.1038/nsmb.2029Received03 September 2010Accepted18 January 2011Published online20 March 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Homologous recombination (also termed homology-directed repair, HDR) is a major pathway for the repair of DNA interstrand cross-links (ICLs) in mammalian cells. Cells from individuals with Fanconi anemia (FA) are characterized by extreme ICL sensitivity, but their reported defect in HDR is mild. Here we examined ICL-induced HDR using a GFP reporter and observed a profound defect in ICL-induced HDR in FA cells, but only when the reporter could replicate. View full text Figures at a glance * Figure 1: ICL induction of HDR in mammalian cells. () HDR reporters. After DNA damage in the upstream GFP repeat, HDR with the downstream GFP repeat leads to a GFP+ gene. A site-specific ICL forms from psoralen conjugated to a TFO (pso-TFO)30. A site-specific DSB is formed by I-SceI endonuclease. () ICL formation in TR-GFP. The TFO binds to the TR-GFP plasmid sequence forming a triplex. Psoralen intercalates at the TpA site and after UVA exposure forms an ICL, blocking DraI cleavage. Lower-case nucleotides within the TFO are locked nucleic acids (LNA), which enhance the stability of the triplex in vivo11; all cytosines are methylated at position 5 (italics). () DraI protection assay to quantify in vivo ICL formation. DNA was isolated from U2OS cells transfected with pso-TFO:TR-GFP and treated with UVA. Controls were (left) unconjugated TFO and (right) a conjugated TFO that cannot bind TR-GFP (pso-mTFO). ICL formation is distinguished from noncovalent triplex DNA formation by addition of an oligonucleotide complementary to th! e TFO before DraI cleavage; this binds to the TFO released from the triplex upon heating (Supplementary Fig. 1d). The 3.5-kb fragment is indicative of ICL formation (arrow). Thin bar, probe. () ICL-induced HDR in U2OS cells. Triplexes formed in vitro were transfected into U2OS cells that were then treated with UVA. GFP+ cells arise from HDR of the ICL in TR-GFP and are significantly less abundant without UVA or pso-TFO (P = 0.0003, unpaired t-test). Transfection efficiency is monitored by co-transfection of a DsRed expression vector. Error bars, ± 1 s.d.; n = 4 replicates from two independent experiments. () ICL-induced HDR, like DSB-induced HDR, is significantly reduced in Brca2 mutant V-C8 cells compared with BRCA2-complemented V-C8 cells (P < 0.0001). Triplexes formed between TR-GFP and either pso-TFO (pso) or an unmodified TFO (+) were transfected into cells that were then treated with UVA. For DSB-induced HDR, DR-GFP was transfected into cells with or without the I-Sc! eI expression vector. n = 5 replicates from two independent ex! periments. * Figure 2: ICL- and DSB-induced HDR in mouse ES cell mutants. Percentage GFP+ cells for each mutant was normalized to the indicated control. (–) BRCA1-deficient cells have substantially reduced ICL- and DSB-induced HDR (); Ku70-deficient cells have only increased DSB-induced HDR (); ERCC1-deficient cells have only reduced ICL-induced HDR (); FANCA-deficient cells have mildly reduced ICL- and DSB-induced HDR (). For ICL-induced HDR, cells were transfected with pso-TFO:TR-GFP or TFO:TR-GFP and then treated with UVA. Error bars, ± 1 s.d. See Supplementary Table 1 for statistical analysis. * Figure 3: FANCA-dependent, replication-coupled ICL-induced HDR. () HDR reporters with EBV OriP for replication in EBNA1-expressing cells. (–) HDR assays in U2OS cells stably expressing EBNA1 (+) or not (−). DSB-induced HDR is only moderately increased with EBNA1 and OriP (), whereas ICL-induced HDR is substantially increased with EBNA1 plus OriP from either in vivo () or in vitro ICL formation without () or with () removal of the TFO tail. All comparisons of I-SceI or pso-TFO with or without EBNA1 plus OriP have P ≤ 0.0002. Error bars, ± 1 s.d.; n = 4 or 5 replicates from two independent experiments. (–) HDR assays in GM6914 or FANCA-complemented GM6914 cells expressing EBNA1 (+) or not (−). DSB-induced HDR is moderately increased with FANCA and EBNA1 plus OriP (). With EBNA1, ± FANCAP = 0.0001; without EBNA1, ± FANCAP = 0.005; with FANCA, ± EBNA1P = 0.004; without FANCA, ± EBNA1P = 0.26. n = 4 replicates from two independent experiments. By contrast, ICL-induced HDR is substantially increased with FANCA and EBNA1 plus Ori! P from either in vivo () or in vitro ICL formation without () or with () removal of the TFO tail. With EBNA1 and FANCA, P < 0.0001 compared with any other condition. Error bars, ± 1 s.d.; n = 4–6 replicates from two or three independent experiments (,); n = 3 replicates from one experiment (). For in vivo ICL formation, TR-OriP-GFP bound to the indicated triplex was transfected into cells that were then treated with UVA. For in vitro TFO formation, TR-OriP-GFP was bound to the indicated triplex and then exposed to UVA before transfection. () Model for ICL repair by HDR. ICLs stall converging replication forks. Incisions on both sides of the ICL create strand breaks, and translesion synthesis (TLS) allows replication past the lesion. The adduct is removed, and strand invasion leads to HDR. As replication from OriP has been reported to be predominately unidirectional29, HDR in TR-GFP likely proceeds after stalling of a single replication fork. Author information * Author information * Supplementary information Affiliations * Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. * Koji Nakanishi, * Erika Brunet & * Maria Jasin * Department of Public Health and Cell Biology, Section of Anatomy, University of Rome Tor Vergata, Rome, Italy. * Francesca Cavallo & * Marco Barchi * Muséum National d'Histoire Naturelle, Paris, France. * Loïc Perrouault, * Carine Giovannangeli & * Erika Brunet * Centre National de la Recherche Scientifique UMR7196, Paris, France. * Loïc Perrouault, * Carine Giovannangeli & * Erika Brunet * INSERM U565, Paris, France. * Loïc Perrouault, * Carine Giovannangeli & * Erika Brunet * Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. * Mary Ellen Moynahan Contributions K.N. designed and performed experiments, analyzed data and assisted in manuscript preparation; F.C. performed experiments and analyzed data; L.P. prepared the ICL-only substrates; C.G. supervised L.P. and assisted in manuscript preparation; M.E.M. provide supervision, analyzed data and assisted in manuscript preparation; M.B. supervised F.C. and contributed to data analysis; E.B. designed experiments and assisted in manuscript preparation; M.J. designed the experiments, supervised the project and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Maria Jasin Author Details * Koji Nakanishi Search for this author in: * NPG journals * PubMed * Google Scholar * Francesca Cavallo Search for this author in: * NPG journals * PubMed * Google Scholar * Loïc Perrouault Search for this author in: * NPG journals * PubMed * Google Scholar * Carine Giovannangeli Search for this author in: * NPG journals * PubMed * Google Scholar * Mary Ellen Moynahan Search for this author in: * NPG journals * PubMed * Google Scholar * Marco Barchi Search for this author in: * NPG journals * PubMed * Google Scholar * Erika Brunet Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Jasin Contact Maria Jasin Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (776K) Supplementary Methods, Supplementary Figures 1–4 and Supplementary Table 1 Additional data
• Structural organization of brain-derived mammalian prions examined by hydrogen-deuterium exchange
  - Nat Struct Mol Biol 18(4):504-506 (2011)
  Nature Structural & Molecular Biology | Brief Communication Structural organization of brain-derived mammalian prions examined by hydrogen-deuterium exchange * Vytautas Smirnovas1, 3 * Gerald S Baron2, 3 * Danielle K Offerdahl2 * Gregory J Raymond2 * Byron Caughey2 * Witold K Surewicz1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:504–506Year published:(2011)DOI:doi:10.1038/nsmb.2035Received22 July 2010Accepted20 January 2011Published online27 March 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg One of the mysteries in prion research is the structure of the infectious form of mammalian prion protein PrPSc. Here we used mass spectrometry analysis of hydrogen-deuterium exchange to examine brain-derived PrPSc. Our data indicate that, contrary to popular models, prion-protein conversion involves refolding of the entire region from residue ~80–90 to the C-terminus, which in PrPSc consists of β-strands and relatively short turns and/or loops, with no native α-helices present. View full text Figures at a glance * Figure 1: Deuterium incorporation for peptic fragments derived from different types of misfolded prion-protein aggregates. () PrPSc from wild-type mice infected with the 22L strain of scrapie after 240 h of exchange. () PrPSc from transgenic GPI− mice infected with the 22L strain of scrapie after 5 min and after 240 h of exchange. () PrPSc from transgenic GPI− mice infected with the Chandler strain of scrapie after 240 h of exchange. () PrPSc from transgenic GPI− mice infected with the ME7 strain of scrapie after 240 h of exchange. () Amyloid fibrils formed from the recombinant mouse prion-protein residues 89–231 after 240 h of exchange. Error bars indicate s.d. (3–5 experiments). * Figure 2: Schematic representation of prion-protein secondary structure in the β-helix (B) and spiral (S) models of PrPSc. Black arrows, curved lines and solid lines represent β-strands, α-helices and loops or unordered segments, respectively. Color bars immediately below the sequence represent the percentage of deuterium incorporation for GPI− 22L PrPSc after 240 h of exchange. * Figure 3: Pairwise comparison of difference in deuterium incorporation after 240‐h exchange for different PrPSc strains. () 22L and Chandler GPI−PrPSc. () 22L and ME7 GPI−PrPSc. () Chandler and ME7 GPI−PrPSc. Data are based on 3–5 experiments using two different preparations of 22L and Chandler GPI−PrPSc and a single preparation of ME7 GPI−PrPSc. Error bars indicate s.d. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this study. * Vytautas Smirnovas & * Gerald S Baron Affiliations * Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio, USA. * Vytautas Smirnovas & * Witold K Surewicz * Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA. * Gerald S Baron, * Danielle K Offerdahl, * Gregory J Raymond & * Byron Caughey Contributions V.S. conducted and analyzed H/D exchange experiments. G.J.R. and D.K.O. did all animal-associated work, from animal inoculations to dissection of brain tissue. G.S.B., D.K.O. and G.J.R. prepared PrPSc samples and carried out their biochemical characterization. B.C. did FTIR experiments. W.K.S. wrote the manuscript and coordinated the entire project. G.S.B., V.S. and B.C. discussed the results and revised the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Witold K Surewicz Author Details * Vytautas Smirnovas Search for this author in: * NPG journals * PubMed * Google Scholar * Gerald S Baron Search for this author in: * NPG journals * PubMed * Google Scholar * Danielle K Offerdahl Search for this author in: * NPG journals * PubMed * Google Scholar * Gregory J Raymond Search for this author in: * NPG journals * PubMed * Google Scholar * Byron Caughey Search for this author in: * NPG journals * PubMed * Google Scholar * Witold K Surewicz Contact Witold K Surewicz Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–4, Supplementary Methods and Supplementary Discussion Additional data
• An effect of DNA sequence on nucleosome occupancy and removal
  - Nat Struct Mol Biol 18(4):507-509 (2011)
  Nature Structural & Molecular Biology | Brief Communication An effect of DNA sequence on nucleosome occupancy and removal * Xin Wang1, 2 * Gene O Bryant1, 2 * Monique Floer1 * Dan Spagna1 * Mark Ptashne1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:507–509Year published:(2011)DOI:doi:10.1038/nsmb.2017Received24 May 2010Accepted13 January 2011Published online06 March 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg A barrier phases nucleosomes at the yeast (Saccharomyces cerevisiae) GAL1–GAL10 genes. Here we separate nucleosome positioning from occupancy and show that the degree of occupancy of these phased sites is predictably determined by the underlying DNA sequences. As this occupancy is increased (by sequence alteration), nucleosome removal upon induction is decreased, as is mRNA production. These results explain why promoter sequences have evolved to form nucleosomes relatively inefficiently. View full text Figures at a glance * Figure 1: Chromatin architecture at the GAL1–GAL10 locus before and after induction. () The wild-type locus. The distribution of nucleosomes before induction, as well as the fractional occupancy of each implied site, is indicated in blue, and the corresponding values following induction are shown in yellow. The data was obtained using the assay of Bryant et al.2. The UASg bears four Gal4-binding sites shown in cyan; the RSC–nucleosome complex is indicated by a cyan oval. Nucleosomes flanking the UASg are shown as green ovals. The shaded areas −1 and −2 indicate the sites of positioned (phased) nucleosomes in the GAL1 promoter. The 5′ ends of the GAL1 and GAL10 genes are shown as horizontal black bars, and the GAL1 TATA box by the small vertical blue bar that lies between sites −1 and −2. The distribution shown in blue is unchanged by addition or deletion of the activator Gal4, but nucleosome removal, as shown in yellow, requires Gal4 and the inducer galactose (2% in this case, added to cells growing in raffinose). The envelopes encompassing the c! urves indicate the range of experimental error, calculated as described by Bryant et al.2. () As in except that the superbinder sequence of Supplementary Table 1 has been substituted for the wild-type sequence at site −1, as indicated by the horizontal magenta bar. () As in except that the superbinder sequence has been substituted for the wild-type sequence at site −2. * Figure 2: The effects of increasing nucleosome-forming propensities on nucleosome occupancies at site −1. () The bottom line indicates the positions of GC and AT/TT/TA dinucleotide elements in a 133-bp sequence designed to form nucleosomes with high efficiency. Each successive TA element is separated by 10 bp, as is each successive GC element. At the top is the array of these sequence elements found in the wild-type (WT) sequence at position −1 in Figure 1. Each successive sequence (starting at the top) was modified by sequential substitutions of 20 bp, resulting in the distribution of TA and GC elements as indicated. The predicted nucleosome-forming propensities of these sequences increase from top to bottom (see text). () Nucleosome occupancies were determined, before (blue) and some 30 min after (yellow) induction, for wild type (WT), superbinder and five other sequences (M1–M5) substituted at site −1 (see ). * Figure 3: Nucleosome removal and mRNA production. () Cells bearing the substitution mutant of Figure 1c (which bears the superbinder at site −2) and wild-type (WT) cells, growing in 2% glucose, were transferred to medium containing 0.1% glucose and 2% galactose. Nucleosome occupancy at the WT site −2 (yellow) and the superbinder-substituted site −2 (blue) was assayed at the indicated time points after induction. () Aliquots of cells used for the experiment of were assayed for GAL1 mRNA using QPCR as described1. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Xin Wang & * Gene O Bryant Affiliations * Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA. * Xin Wang, * Gene O Bryant, * Monique Floer, * Dan Spagna & * Mark Ptashne Contributions X.W., G.O.B. and M.P. designed the experiments. X.W., G.O.B. and D.S. performed the experiments. X.W. and G.O.B. analyzed the data. X.W., G.O.B., M.F. and M.P. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Mark Ptashne Author Details * Xin Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Gene O Bryant Search for this author in: * NPG journals * PubMed * Google Scholar * Monique Floer Search for this author in: * NPG journals * PubMed * Google Scholar * Dan Spagna Search for this author in: * NPG journals * PubMed * Google Scholar * Mark Ptashne Contact Mark Ptashne Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (292K) Supplementary Figure 1, Supplementary Table 1 and Supplementary Methods Additional data
• Impact of chromatin structure on sequence variability in the human genome
  - Nat Struct Mol Biol 18(4):510-515 (2011)
  Nature Structural & Molecular Biology | Analysis Impact of chromatin structure on sequence variability in the human genome * Michael Y Tolstorukov1, 2 * Natalia Volfovsky3 * Robert M Stephens3 * Peter J Park1, 2, 4 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:510–515Year published:(2011)DOI:doi:10.1038/nsmb.2012Received02 December 2009Accepted10 December 2010Published online13 March 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 DNA sequence variations in individual genomes give rise to different phenotypes within the same species. One mechanism in this process is the alteration of chromatin structure due to sequence variation that influences gene regulation. We composed a high-confidence collection of human single-nucleotide polymorphisms and indels based on analysis of publicly available sequencing data and investigated whether the DNA loci associated with stable nucleosome positions are protected against mutations. We addressed how the sequence variation reflects the occupancy profiles of nucleosomes bearing different epigenetic modifications on genome scale. We found that indels are depleted around nucleosome positions of all considered types, whereas single-nucleotide polymorphisms are enriched around the positions of bulk nucleosomes but depleted around the positions of epigenetically modified nucleosomes. These findings indicate an increased level of conservation for the sequences associated ! with epigenetically modified nucleosomes, highlighting complex organization of the human chromatin. View full text Figures at a glance * Figure 1: Genome-wide distributions of indel and SNP events. (,) Distributions of indel () and SNP () frequencies around stable nucleosome positions. Results are shown for a combined set of nucleosome positions (gray) and for individual nucleosome sets: bulk (cyan), H2A.Z (blue) and H3K4me3 (red). The frequency profiles were normalized and smoothed as described in Online Methods. In each plot, black dashed vertical line at position zero and red dashed vertical lines at positions ± 73 bp refer to nucleosome center and nucleosomal size, respectively. (,) Autocorrelation profiles for indel () and SNP () occurrences. Thin gray lines correspond to the initial profile calculated with 1-bp lag increments, and thick red line represents loess smoothing of the initial data. Two local maxima in the indel profile corresponding to mono- and dinucleosomal sizes are indicated with numbers. * Figure 2: Distribution of indels (red), SNPs (green) and stable nucleosome (nuc) positions from combined set (black) around boundaries between introns and exons. () Intron-exon boundaries. () Exon-intron boundaries. Zero position in each plot (black dashed vertical line) corresponds to position of boundary. Exonic coordinates were taken from the USCS track RefGene for known protein-coding genes from the NCBI messenger RNA sequences collection (RefSeq)43, 44. First and last exons were excluded from the analysis. Only genes for which no alternative start site was reported were considered in this analysis (14,946 genes). The combined nucleosome set (all nucs) was used to produce this plot. The frequency profiles were calculated as described in Online Methods. Heat maps shown in the bottom panels represent detrended profiles where large-scale variations were removed. * Figure 3: Distribution of indels (red), SNPs (green) and nucleosome positions (black) around TSSs and TESs of human genes. Profiles were calculated as described in Online Methods. As in Figure 2, heat maps shown in the bottom panels represent detrended profiles. () Profiles around TSSs (position zero, dashed black vertical line). The combined set of stable nucleosome positions (all nucs) was used. Genes were oriented in the direction of transcription in such a way that the upstream region is shown on the left of the TSS and the downstream region is shown on the right. () Profiles are shown separately for the frequency of indels (solid and dotted red lines) and bulk nucleosome occupancy (solid and dotted black lines) for the subsets of genes associated and not associated with CpG islands at TSSs. Solid and dotted black ovals represent nucleosomes at position +1 in CpGi and non-CpGi genes and are shown for a nucleosome size reference. Coordinates of CpG islands were taken from USCS genome browser annotation43. () Profiles computed around TESs (position zero, black dashed vertical line) for all gen! es. The combined-nucleosome set was used. * Figure 4: Distribution of SNP frequencies around stable nucleosome positions. (–) SNP frequency distributions in the regions that are proximal () and distal () to the TSSs of human genes and around nucleosome positions located within coding regions (). TSS-proximal and TSS-distant nucleosome positions were identified as those located less than 1 kb and more than 2 kb from the closest TSS, respectively. Coding regions are defined according to the annotation of the USCS genome browser43. Normalized profiles are shown for the positions from the combined-nucleosome set (gray) and for the individual-nucleosome sets: bulk (cyan), H2A.Z (blue) and H3K4me3 (red). Vertical dashed lines at zero and ± 73 bp refer to the nucleosome position and size. * Figure 5: Interplay of chromatin-mediated mutation bias and selection can shape sequence variation profiles (compare to schematic illustration in ref. 38). () Bulk and epigenetically modified nucleosomes are shown with blue and red ovals, respectively. Green and orange lines represent major effects of nucleosome presence on the mutation rates of SNPs and indels, respectively, and black line represents selection pressure acting on the DNA sequence. () The difference in the indel rate inside and outside nucleosomes mainly determines the indel frequency profile observed in the genome (orange), whereas the SNP frequency profile (green) is mainly affected by selection. Author information * Abstract * Author information * Supplementary information Affiliations * Center for Biomedical Informatics, Harvard Medical School, Boston, Massachusetts, USA. * Michael Y Tolstorukov & * Peter J Park * Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts, USA. * Michael Y Tolstorukov & * Peter J Park * Advanced Biomedical Computing Center, Information Systems Program, SAIC-Frederick, National Cancer Institute at Frederick, Frederick, Maryland, USA. * Natalia Volfovsky & * Robert M Stephens * HST Informatics Program at Children's Hospital Boston, Boston, Massachusetts, USA. * Peter J Park Contributions M.Y.T. performed all analyses. N.V. and R.M.S. produced the collections of sequence variations. P.J.P. directed the project. M.Y.T. and P.J.P. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Peter J Park Author Details * Michael Y Tolstorukov Search for this author in: * NPG journals * PubMed * Google Scholar * Natalia Volfovsky Search for this author in: * NPG journals * PubMed * Google Scholar * Robert M Stephens Search for this author in: * NPG journals * PubMed * Google Scholar * Peter J Park Contact Peter J Park 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 1–9, Supplementary Tables 1–4 and Supplementary Notes 1 and 2 Additional data
• A direct role for Hsp90 in pre-RISC formation in Drosophila
  - Nat Struct Mol Biol 18(4):516 (2011)
  Nature Structural & Molecular Biology | Corrigendum A direct role for Hsp90 in pre-RISC formation in Drosophila * Tomohiro Miyoshi * Akiko Takeuchi * Haruhiko Siomi * Mikiko C SiomiJournal name:Nature Structural & Molecular BiologyVolume: 18,Page:516Year published:(2011)DOI:doi:10.1038/nsmb0411-516aPublished online06 April 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Struct. Mol. Biol.17, 1024–1026 (2010); published online 18 July 2010; corrected after print 25 August 2010 In the version of this article initially published, "2′-O-methyl group at the 10th nucleotide" should have read "2′-O-methyl groups at the 9th and 10th nucleotides." The error has been corrected in the HTML and PDF versions of the article. Additional data Author Details * Tomohiro Miyoshi Search for this author in: * NPG journals * PubMed * Google Scholar * Akiko Takeuchi Search for this author in: * NPG journals * PubMed * Google Scholar * Haruhiko Siomi Search for this author in: * NPG journals * PubMed * Google Scholar * Mikiko C Siomi Search for this author in: * NPG journals * PubMed * Google Scholar
• Structural and functional analyses of minimal phosphopeptides targeting the polo-box domain of polo-like kinase 1
  - Nat Struct Mol Biol 18(4):516 (2011)
  Nature Structural & Molecular Biology | Corrigendum Structural and functional analyses of minimal phosphopeptides targeting the polo-box domain of polo-like kinase 1 * Sang-Moon Yun * Tinoush Moulaei * Dan Lim * Jeong K Bang * Jung-Eun Park * Shilpa R Shenoy * Fa Liu * Young H Kang * Chenzhong Liao * Nak-Kyun Soung * Sunhee Lee * Do-Young Yoon * Yoongho Lim * Dong-Hee Lee * Akira Otaka * Ettore Appella * James B McMahon * Marc C Nicklaus * Terrence R Burke Jr * Michael B Yaffe * Alexander Wlodawer * Kyung S LeeJournal name:Nature Structural & Molecular BiologyVolume: 18,Page:516Year published:(2011)DOI:doi:10.1038/nsmb0411-516bPublished online06 April 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Struct. Mol. Biol.16, 876–882 (2009); published online 13 July 2009; corrected after print 21 September 2010 In the version of this article initially published, two of the numbers shown in Table 1 had the wrong sign. The error has been corrected in the HTML and PDF versions of the article. Additional data Author Details * Sang-Moon Yun Search for this author in: * NPG journals * PubMed * Google Scholar * Tinoush Moulaei Search for this author in: * NPG journals * PubMed * Google Scholar * Dan Lim Search for this author in: * NPG journals * PubMed * Google Scholar * Jeong K Bang Search for this author in: * NPG journals * PubMed * Google Scholar * Jung-Eun Park Search for this author in: * NPG journals * PubMed * Google Scholar * Shilpa R Shenoy Search for this author in: * NPG journals * PubMed * Google Scholar * Fa Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Young H Kang Search for this author in: * NPG journals * PubMed * Google Scholar * Chenzhong Liao Search for this author in: * NPG journals * PubMed * Google Scholar * Nak-Kyun Soung Search for this author in: * NPG journals * PubMed * Google Scholar * Sunhee Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Do-Young Yoon Search for this author in: * NPG journals * PubMed * Google Scholar * Yoongho Lim Search for this author in: * NPG journals * PubMed * Google Scholar * Dong-Hee Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Akira Otaka Search for this author in: * NPG journals * PubMed * Google Scholar * Ettore Appella Search for this author in: * NPG journals * PubMed * Google Scholar * James B McMahon Search for this author in: * NPG journals * PubMed * Google Scholar * Marc C Nicklaus Search for this author in: * NPG journals * PubMed * Google Scholar * Terrence R Burke Jr Search for this author in: * NPG journals * PubMed * Google Scholar * Michael B Yaffe Search for this author in: * NPG journals * PubMed * Google Scholar * Alexander Wlodawer Search for this author in: * NPG journals * PubMed * Google Scholar * Kyung S Lee Search for this author in: * NPG journals * PubMed * Google Scholar
• The C terminus of p53 binds the N-terminal domain of MDM2
  - Nat Struct Mol Biol 18(4):516 (2011)
  Nature Structural & Molecular Biology | Corrigendum The C terminus of p53 binds the N-terminal domain of MDM2 * Masha V Poyurovsky * Chen Katz * Oleg Laptenko * Rachel Beckerman * Maria Lokshin * Jinwoo Ahn * In-Ja L Byeon * Ronen Gabizon * Melissa Mattia * Andrew Zupnick * Lewis M Brown * Assaf Friedler * Carol PrivesJournal name:Nature Structural & Molecular BiologyVolume: 18,Page:516Year published:(2011)DOI:doi:10.1038/nsmb0411-516cPublished online06 April 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Struct. Mol. Biol.17, 982–989 (2010); corrected online 21 September 2010 In the version of this article initially published, the error bars in Figures 1, 2, 4 and 5 were not defined, and a formatting mistake occurred in Supplementary Figure 1a. The errors have been corrected in the PDF and HTML versions of this article. Additional data Author Details * Masha V Poyurovsky Search for this author in: * NPG journals * PubMed * Google Scholar * Chen Katz Search for this author in: * NPG journals * PubMed * Google Scholar * Oleg Laptenko Search for this author in: * NPG journals * PubMed * Google Scholar * Rachel Beckerman Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Lokshin Search for this author in: * NPG journals * PubMed * Google Scholar * Jinwoo Ahn Search for this author in: * NPG journals * PubMed * Google Scholar * In-Ja L Byeon Search for this author in: * NPG journals * PubMed * Google Scholar * Ronen Gabizon Search for this author in: * NPG journals * PubMed * Google Scholar * Melissa Mattia Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew Zupnick Search for this author in: * NPG journals * PubMed * Google Scholar * Lewis M Brown Search for this author in: * NPG journals * PubMed * Google Scholar * Assaf Friedler Search for this author in: * NPG journals * PubMed * Google Scholar * Carol Prives Search for this author in: * NPG journals * PubMed * Google Scholar
• Structure-function analysis of hRPC62 provides insights into RNA polymerase III transcription initiation
  - Nat Struct Mol Biol 18(4):516 (2011)
  Nature Structural & Molecular Biology | Corrigendum Structure-function analysis of hRPC62 provides insights into RNA polymerase III transcription initiation * Stéphane Lefèvre * Hélène Dumay-Odelot * Leyla El-Ayoubi * Aidan Budd * Pierre Legrand * Noël Pinaud * Martin Teichmann * Sébastien FribourgJournal name:Nature Structural & Molecular BiologyVolume: 18,Page:516Year published:(2011)DOI:doi:10.1038/nsmb0411-516dPublished online06 April 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Struct. Mol. Biol.18, 352–358 (2011); published online 27 February 2011; corrected after print 17 March 2011 In the version of this article initially published, reference 20 was not cited in the main text. The error has been corrected in the HTML and PDF versions of the article. Additional data 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 Search for this author in: * NPG journals * PubMed * Google Scholar
• The prospects for designer single-stranded RNA-binding proteins
  - Nat Struct Mol Biol 18(4):516 (2011)
  Nature Structural & Molecular Biology | Erratum The prospects for designer single-stranded RNA-binding proteins * Joel P Mackay * Josep Font * David J SegalJournal name:Nature Structural & Molecular BiologyVolume: 18,Page:516Year published:(2011)DOI:doi:10.1038/nsmb0411-516ePublished online06 April 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Struct. Mol. Biol.18, 256–261 (2011); published online 27 February 2011; corrected after print 17 March 2011 In the version of this article initially published, in several instances "guanidine" should have read "guanine", "uridine" should have read "uracil", and "adenine" should have read adenosine; in two instances; "tetratrispolin" should have read "tristetraprolin"; and Figure 2c,d should have illustrated the structure from PDB record 1M8Y. These errors have been corrected in the HTML and PDF versions of the article. Additional data Author Details * 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 Search for this author in: * NPG journals * PubMed * Google Scholar