The structural basis of nucleic acid recognition by Toll-like receptors

Summary

Principal Investigator: Yorgo Modis
Abstract: DESCRIPTION (provided by applicant): Toll-like receptors (TLRs) generate an innate immune signaling response upon recognizing broadly conserved microbial extracellular structures. Viral RNA is recognized by!TLR3, TLR7 and TLR8, and microbial DNA is recognized by TLR9. Until recently the prevailing paradigm was that TLR9 recognized unmethylated CpG DNA motifs, which are abundant in bacteria but relatively scarce in mammalian DNA. However, recent studies including our own preliminary data suggest that TLR9 binds natural DNA ligands independently of their sequence and methylation state. We propose a comprehensive analysis of the structural properties that allow TLR9 to recognize microbial DNA including sequence, length, duplex content, methylation state, backbone chemistry (phosphodiester versus phosphorothioate), curvature and higher order structure (such as junctions). We show in preliminary studies that DNA curvature-inducing proteins!HMGB1 and histones H2A and H2B significantly enhance binding to the C-terminal cleavage fragment of TLR9, suggesting that TLR9 preferentially recognizes curved DNA backbones. To determine the extent to which DNA curvature alone is responsible for the binding enhancement, we propose to measure the TLR9 binding affinity of DNA minicircles containing 75 to 120 base pairs. Since nucleosomes can induce TLR-dependent auto immunogenic signaling, we propose an in vitro biophysical analysis of whole nucleosomes as TLR9 ligands! These in vitro studies will be validated in vivo by measuring TLR9-dependent signaling responses in cells stimulated with various DNA or protein-DNA ligands including minicircles, nucleosomes, junctions and methylated DNA ligands. The TLR7/8/9 ectodomains must be proteolytically cleaved in order to produce receptors that are capable of signaling. In the first study with cleaved TLR9, we show in our preliminary data that both the N- and C-terminal TLR9 ectodomain fragments participate in ligand binding and receptor dimerization. We therefore hypothesize that the two fragments remain associated after proteolytic cleavage in the endosome, and that cleavage may be necessary for TLR9 to undergo the ligand-induced conformational change that activates the receptor. To test this hypothesis, we will explore the physical and functional relationships between the two TLR9 ectodomain fragments, and elucidate the physical basis of proteolytic activation using biophysical approaches. The lack of structural information for TLR7/8/9 limits our understanding of nucleic acid recognition by these receptors. We propose to use electron cryomicroscopy and X-ray crystallography as complementary approaches to gain insight into the structural basis of TLR9-DNA recognition. By providing a molecular-level understanding of the recognition of microbial DNA by TLR9, this project will provide the necessary tools to create more potent vaccine adjuvants, and a new class of anti-inflammatory therapeutics with a wide range of applications including in particular systemic lupus erythematosus, asthma, septic shock syndrome and organ transplant rejection.
Funding Period: 2012-08-01 - 2016-07-31
more information: NIH RePORT

Top Publications

  1. pmc Selective pressure causes an RNA virus to trade reproductive fitness for increased structural and thermal stability of a viral enzyme
    Moshe Dessau
    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States of America
    PLoS Genet 8:e1003102. 2012
  2. pmc Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses
    Jimin Wang
    Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA Electronic address
    Virology 454:93-101. 2014
  3. pmc A novel membrane fusion protein family in Flaviviridae?
    Yue Li
    Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA
    Trends Microbiol 22:176-82. 2014
  4. pmc Relating structure to evolution in class II viral membrane fusion proteins
    Yorgo Modis
    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA Electronic address
    Curr Opin Virol 5:34-41. 2014
  5. pmc RNA:DNA hybrids are a novel molecular pattern sensed by TLR9
    Rachel E Rigby
    MRC Human Genetics Unit, MRC IGMM University of Edinburgh, Edinburgh, UK
    EMBO J 33:542-58. 2014
  6. pmc Viral membrane fusion and nucleocapsid delivery into the cytoplasm are distinct events in some flaviviruses
    Adel M Nour
    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States of America
    PLoS Pathog 9:e1003585. 2013
  7. pmc Chitinase 3-like 1 regulates cellular and tissue responses via IL-13 receptor α2
    Chuan Hua He
    Section of Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06520 8057, USA
    Cell Rep 4:830-41. 2013
  8. pmc Crystal structure of glycoprotein E2 from bovine viral diarrhea virus
    Yue Li
    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA
    Proc Natl Acad Sci U S A 110:6805-10. 2013
  9. pmc Crystal structure of glycoprotein C from Rift Valley fever virus
    Moshe Dessau
    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA
    Proc Natl Acad Sci U S A 110:1696-701. 2013
  10. ncbi Endosomal vesicles as vehicles for viral genomes
    Adel M Nour
    Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA
    Trends Cell Biol 24:449-54. 2014

Detail Information

Publications10

  1. pmc Selective pressure causes an RNA virus to trade reproductive fitness for increased structural and thermal stability of a viral enzyme
    Moshe Dessau
    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States of America
    PLoS Genet 8:e1003102. 2012
    ....
  2. pmc Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses
    Jimin Wang
    Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA Electronic address
    Virology 454:93-101. 2014
    ..The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. ..
  3. pmc A novel membrane fusion protein family in Flaviviridae?
    Yue Li
    Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA
    Trends Microbiol 22:176-82. 2014
    ..This and other recently identified structural relationships between viral fusion proteins shift the paradigm for how these proteins evolved. ..
  4. pmc Relating structure to evolution in class II viral membrane fusion proteins
    Yorgo Modis
    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA Electronic address
    Curr Opin Virol 5:34-41. 2014
    ..The structural relationships of newly identified class II proteins, reviewed herein, shift the paradigm for how these proteins evolved. ..
  5. pmc RNA:DNA hybrids are a novel molecular pattern sensed by TLR9
    Rachel E Rigby
    MRC Human Genetics Unit, MRC IGMM University of Edinburgh, Edinburgh, UK
    EMBO J 33:542-58. 2014
    ..Hybrids therefore represent a novel molecular pattern sensed by the innate immune system and so could play an important role in host response to viruses and the pathogenesis of autoimmune disease. ..
  6. pmc Viral membrane fusion and nucleocapsid delivery into the cytoplasm are distinct events in some flaviviruses
    Adel M Nour
    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States of America
    PLoS Pathog 9:e1003585. 2013
    ..Virus entry modulates intracellular calcium release and phosphatidylinositol-3-phosphate kinase signaling. Moreover, the broadly cross-reactive therapeutic antibody scFv11 binds to virus-like particles and inhibits fusion. ..
  7. pmc Chitinase 3-like 1 regulates cellular and tissue responses via IL-13 receptor α2
    Chuan Hua He
    Section of Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06520 8057, USA
    Cell Rep 4:830-41. 2013
    ..Thus, IL-13Rα2 is a GH 18 receptor that plays a critical role in Chi3l1 effector responses. ..
  8. pmc Crystal structure of glycoprotein E2 from bovine viral diarrhea virus
    Yue Li
    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA
    Proc Natl Acad Sci U S A 110:6805-10. 2013
    ..From the structure of E2, we propose alternative possible membrane fusion mechanisms. We expect the pestivirus fusion apparatus to be conserved in hepatitis C virus...
  9. pmc Crystal structure of glycoprotein C from Rift Valley fever virus
    Moshe Dessau
    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA
    Proc Natl Acad Sci U S A 110:1696-701. 2013
    ..Unanticipated similarities between G(C) and flavivirus envelope proteins reveal an evolutionary link between the two virus families and provide insights into the organization of G(C) in the outer shell of RVFV...
  10. ncbi Endosomal vesicles as vehicles for viral genomes
    Adel M Nour
    Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA
    Trends Cell Biol 24:449-54. 2014
    ..When secreted as exosomes, ILVs containing viral genomes can infect permissive cells or activate immune responses in myeloid cells. We therefore propose that endosomal ILVs and exosomes are key effectors of viral pathogenesis. ..

Research Grants30

  1. Host-pathogen competition in IFN mediated antiviral defense
    Jae U Jung; Fiscal Year: 2013
    ....
  2. Transplant Tolerance in Non-Human Primates
    STUART JOHNSTON KNECHTLE; Fiscal Year: 2013
    ..This goal will be accomplished via four interrelated projects and two supporting cores. ..
  3. IRAK4 and systemic lupus erythematosus
    Andrei E Medvedev; Fiscal Year: 2013
    ..These advances would be of key importance for basic immunology of SLE, and for improving public health of lupus patients in the U.S. ..
  4. Toll-Like Receptors in Systemic Autoimmune Disease
    Ann Marshak-Rothstein; Fiscal Year: 2013
    ....
  5. Pacific NorthWest Regional Center of Excellence (PNWRCE)
    Jay A Nelson; Fiscal Year: 2013
    ..pseudomallei host pathogen response during both the septicemic as well as the intracellular phases of the disease. ..
  6. Southeast Regional Centers of Excellence for Biodefense &Emerging Infectious Di
    Philip Frederick Sparling; Fiscal Year: 2013
    ..SERCEB brings new investigators to the biodefense effort through a combination of educational programs, support of innovative new projects, and the synergistic interactions among its world-class investigators. ..
  7. New England Regional Center of Excellence in Biodefense and Emerging Infectious D
    Dennis L Kasper; Fiscal Year: 2013
    ..NERCE will also continue its Developmental Projects program and Career Development in Biodefense program in an effort to initiate new research efforts and to attract new investigators to this field. ..
  8. Northeast Biodefense Center
    W Ian Lipkin; Fiscal Year: 2013
    ..As a Center based in a School of Public Health and a State Department of Health, the NBC has a firm commitment to and practical understanding of Emergency Preparedness. ..
  9. Molecular Analyses and Interventions for Biodefense and Emerging Pathogens
    Olaf Schneewind; Fiscal Year: 2013
    ..Research and training at the GLRCE is governed by a mechanism involving ongoing review of scientific excellence and translational goals, inter-institutional advisory boards and external scientific advisory bodies. ..
  10. Resolution Mechanisms in Acute Inflammation: Resolution Pharmacology
    CHARLES NICHOLAS SERHAN; Fiscal Year: 2013
    ..Selected synthetic SPM will be scaled-up for demonstration of their unique mode of action in vivo in a resolution pharmacology core using experimental disease models. Our broad goal is to bring forth new treatments in resolution. ..
  11. Characterizing DExD/H box helicases as viral sensors in human dendritic cells
    Yong Jun Liu; Fiscal Year: 2013
    ....
  12. Toll-like receptor 9 proteolytic processing and signaling
    Cynthia A Leifer; Fiscal Year: 2013
    ..But more importantly, will determine whether proteolysis is, in fact, a promising new pathway for drug development, or of lesser significance compared with other regulatory mechanisms. ..
  13. Rocky Mountain Regional Center of Excellence or Biodefense and Emerging Infectiou
    John T Belisle; Fiscal Year: 2013
    ..abstract_text> ..
  14. Electron Microscopy of Biological Macromolecules
    Kenneth H Downing; Fiscal Year: 2013
    ....
  15. Protein homeostasis mechanisms underlying enterovirus replication and evolution
    Raul Andino; Fiscal Year: 2013
    ..Core A: Administrative Core;and Core B: "High-throughput functional genomics and proteomics core. ..
  16. Structural basis of TLR and NLR recognition and signaling
    Ian A Wilson; Fiscal Year: 2013
    ..Andrew Ward and David Baker will complement our biophysical and functional studies as well as stimulate development of agonists and antagonists as therapeutics in compromised immunity or in hyper- inflammatory diseases. ..