Abstract
Microbial and synthetic DNA rich in CpG dinucleotides stimulates Toll-like receptor 9 (TLR9), whereas DNA lacking CpG either is inert or can inhibit TLR9 activation. The molecular mechanisms by which TLR9 becomes activated or is inhibited are not well understood. Here we show that TLR9 bound to stimulatory and inhibitory DNA; however, only stimulatory DNA led to substantial conformational changes in the TLR9 ectodomain. In the steady state, 'inactive' TLR9 homodimers formed in an inactivated conformation. Binding of DNA containing CpG, but not of DNA lacking CpG, to TLR9 dimers resulted in allosteric changes in the TLR9 cytoplasmic signaling domains. In endosomes, conformational changes induced by DNA containing CpG resulted in close apposition of the cytoplasmic signaling domains, a change that is probably required for the recruitment of signaling adaptor molecules. Our results indicate that the formation of TLR9 dimers is not sufficient for its activation but instead that TLR9 activation is regulated by conformational changes induced by DNA containing CpG.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout





Similar content being viewed by others
Change history
19 October 2007
In the version of this article initially published, the distance values reported in Tables 1 and 2 are incorrect. The correct values are provided in the revised tables. Accordingly, line 6 on p 774 should read “7.3 nm”; line 11 on p 774 should read “a 12% decrease”; line 12 on p 777 should read “7.0 nm”; and lines 17–20 on p 777 should read “We calculated the C-terminal intermolecular distance in the endosome to be less 5.4 nm. Given the fact that the donor and acceptor fluorophores are buried inside the fluorescent proteins, their minimal distance is approximately 5.0 nm. Thus, these measurements indicate that the TLR9 TIR domains were brought in close proximity after the binding of CpG DNA ligand to the TLR9 ectodomains (Table 2).” The error has been corrected in the HTML and PDF versions of the article.
References
Akira, S. & Takeda, K. Toll-like receptor signalling. Nat. Rev. Immunol. 4, 499–511 (2004).
Alexopoulou, L., Holt, A.C., Medzhitov, R. & Flavell, R.A. Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3. Nature 413, 732–738 (2001).
Heil, F. et al. Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science 303, 1526–1529 (2004).
Diebold, S.S., Kaisho, T., Hemmi, H., Akira, S. & Reis e Sousa, C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303, 1529–1531 (2004).
Hemmi, H. et al. A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745 (2000).
Latz, E. et al. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat. Immunol. 5, 190–198 (2004).
Krieg, A.M. et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374, 546–549 (1995).
Means, T.K. et al. Human lupus autoantibody-DNA complexes activate DCs through cooperation of CD32 and TLR9. J. Clin. Invest. 115, 407–417 (2005).
Rifkin, I.R., Leadbetter, E.A., Busconi, L., Viglianti, G. & Marshak-Rothstein, A. Toll-like receptors, endogenous ligands, and systemic autoimmune disease. Immunol. Rev. 204, 27–42 (2005).
Marshak-Rothstein, A. Toll-like receptors in systemic autoimmune disease. Nat. Rev. Immunol. 6, 823–835 (2006).
Krieg, A.M. Therapeutic potential of Toll-like receptor 9 activation. Nat. Rev. Drug Discov. 5, 471–484 (2006).
Agrawal, S. & Kandimalla, E.R. Medicinal chemistry and therapeutic potential of CpG DNA. Trends Mol. Med. 8, 114–121 (2002).
Gay, N.J., Gangloff, M. & Weber, A.N. Toll-like receptors as molecular switches. Nat. Rev. Immunol. 6, 693–698 (2006).
Yasuda, K. et al. CpG motif-independent activation of TLR9 upon endosomal translocation of “natural” phosphodiester DNA. Eur. J. Immunol. 36, 431–436 (2006).
Hartmann, G. & Krieg, A.M. Mechanism and function of a newly identified CpG DNA motif in human primary B cells. J. Immunol. 164, 944–953 (2000).
Ashman, R.F., Goeken, J.A., Drahos, J. & Lenert, P. Sequence requirements for oligodeoxyribonucleotide inhibitory activity. Int. Immunol. 17, 411–420 (2005).
Duramad, O. et al. Inhibitors of TLR-9 act on multiple cell subsets in mouse and man in vitro and prevent death in vivo from systemic inflammation. J. Immunol. 174, 5193–5200 (2005).
Barrat, F.J. et al. Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus. J. Exp. Med. 202, 1131–1139 (2005).
Kelly, S.M., Jess, T.J. & Price, N.C. How to study proteins by circular dichroism. Biochim. Biophys. Acta 1751, 119–139 (2005).
Bell, J.K. et al. The molecular structure of the Toll-like receptor 3 ligand-binding domain. Proc. Natl. Acad. Sci. USA 102, 10976–10980 (2005).
Choe, J., Kelker, M.S. & Wilson, I.A. Crystal structure of human Toll-like receptor 3 (TLR3) ectodomain. Science 309, 581–585 (2005).
Bell, J.K. et al. Leucine-rich repeats and pathogen recognition in Toll-like receptors. Trends Immunol. 24, 528–533 (2003).
Wallrabe, H. & Periasamy, A. Imaging protein molecules using FRET and FLIM microscopy. Curr. Opin. Biotechnol. 16, 19–27 (2005).
Livnah, O. et al. Crystallographic evidence for preformed dimers of erythropoietin receptor before ligand activation. Science 283, 987–990 (1999).
Remy, I., Wilson, I.A. & Michnick, S.W. Erythropoietin receptor activation by a ligand-induced conformation change. Science 283, 990–993 (1999).
de Vos, A.M., Ultsch, M. & Kossiakoff, A.A. Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 255, 306–312 (1992).
Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).
Ghosh, I., Hamilton, A.D. & Regan, L. Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J. Am. Chem. Soc. 122, 5658–5659 (2000).
Jeong, J. et al. Monitoring of conformational change in maltose binding protein using split green fluorescent protein. Biochem. Biophys. Res. Commun. 339, 647–651 (2006).
Hacker, H. et al. Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6. J. Exp. Med. 192, 595–600 (2000).
Ahmad-Nejad, P. et al. Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur. J. Immunol. 32, 1958–1968 (2002).
Krug, A. et al. Identification of CpG oligonucleotide sequences with high induction of IFN-α/β in plasmacytoid dendritic cells. Eur. J. Immunol. 31, 2154–2163 (2001).
Hartmann, G. et al. Rational design of new CpG oligonucleotides that combine B cell activation with high IFN-α induction in plasmacytoid dendritic cells. Eur. J. Immunol. 33, 1633–1641 (2003).
Taylor, I.A., Davis, K.G., Watts, D. & Kneale, G.G. DNA-binding induces a major structural transition in a type I methyltransferase. EMBO J. 13, 5772–5778 (1994).
Weiss, M.A. et al. Folding transition in the DNA-binding domain of GCN4 on specific binding to DNA. Nature 347, 575–578 (1990).
Winkler, F.K. Structure and function of restriction endonucleases. Curr. Opin. Struct. Biol. 2, 93–99 (1992).
Cheng, X., Kumar, S., Posfai, J., Pflugrath, J.W. & Roberts, R.J. Crystal structure of the HhaI DNA methyltransferase complexed with S-adenosyl-L-methionine. Cell 74, 299–307 (1993).
Weber, A.N., Morse, M.A. & Gay, N.J. Four N-linked glycosylation sites in human toll-like receptor 2 cooperate to direct efficient biosynthesis and secretion. J. Biol. Chem. 279, 34589–34594 (2004).
O'Neil, K.T., Hoess, R.H. & DeGrado, W.F. Design of DNA-binding peptides based on the leucine zipper motif. Science 249, 774–778 (1990).
Talanian, R.V., McKnight, C.J. & Kim, P.S. Sequence-specific DNA binding by a short peptide dimer. Science 249, 769–771 (1990).
Weber, A.N. et al. Binding of the Drosophila cytokine Spatzle to Toll is direct and establishes signaling. Nat. Immunol. 4, 794–800 (2003).
Bell, J.K., Askins, J., Hall, P.R., Davies, D.R. & Segal, D.M. The dsRNA binding site of human Toll-like receptor 3. Proc. Natl. Acad. Sci. USA 103, 8792–8797 (2006).
Jiang, Z. et al. Details of Toll-like receptor:adapter interaction revealed by germ-line mutagenesis. Proc. Natl. Acad. Sci. USA 103, 10961–10966 (2006).
Hemmi, H. et al. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat. Immunol. 3, 196–200 (2002).
Lee, J. et al. Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of Toll-like receptor 7. Proc. Natl. Acad. Sci. USA 100, 6646–6651 (2003).
Gorden, K.K. et al. Oligodeoxynucleotides differentially modulate activation of TLR7 and TLR8 by imidazoquinolines. J. Immunol. 177, 8164–8170 (2006).
Acknowledgements
We thank S. Young (Leica Microsystems) and W. Becker (Becker & Hickl) for advice on FLIM measurements. Double-stranded RNA (enhanced GFP small interfering RNA duplex) was from S. Bauer (University of Marburg). Supported by the National Institutes of Health (R01AI065483 and RO1GM54060 to D.T.G and E.L.) and the Norwegian Research Council (T.E. and D.K.).
Author information
Authors and Affiliations
Contributions
E.L., C.M.S, T.E. and D.T.G. wrote the paper; B.G.M. and A.V. cloned expression constructs; E.L., A.V., C.M.S., D.C.G.K. and M.G. did imaging and biochemical analysis; C.J.M. assisted with circular dichroism experiments; W.P.D. assisted with bimolecular fluorescence complementation experiments; M.S.L. helped with the AlphaScreen assay; and all authors discussed experimental results.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Fig. 1
Size exclusion chromatography of TLR–Fc proteins. (PDF 268 kb)
Supplementary Fig. 2
HEK293 cells stably expressing TLR9–YFP (red), TLR9–CFP (green) alone or TLR9–CFP and TLR9–YFP (yellow) together were co-cultured on glass-bottom tissue culture dishes and analyzed by sequential scanning confocal microscopy (left). (PDF 1482 kb)
Rights and permissions
About this article
Cite this article
Latz, E., Verma, A., Visintin, A. et al. Ligand-induced conformational changes allosterically activate Toll-like receptor 9. Nat Immunol 8, 772–779 (2007). https://doi.org/10.1038/ni1479
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ni1479