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Ligand-induced conformational changes allosterically activate Toll-like receptor 9

Nature Immunology volume 8, pages 772–779 (2007)Cite this article

A Corrigendum to this article was published on 01 November 2007

This article has been updated

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.

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Figure 1: TLR9 binds to stimulatory and inhibitory CpG DNA.
Figure 2: Binding of DNA induces distinct conformational changes in TLR9-Fc.
Figure 3: TLR9 is expressed as a preformed homodimer.
Figure 4: Binding of ligand to TLR9 facilitates proximity-assisted folding of the 'split' GFP constructs N-GFP–TLR9 and C-GFP–TLR9 in endosomes.
Figure 5: CpG DNA leads to increased FRET efficiency between CFP-TLR9 and YFP-TLR9 in endosomes.

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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.

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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.).

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Authors and Affiliations

  1. Department of Medicine, Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, 01605, Massachusetts, USA

    Eicke Latz, Anjali Verma, Alberto Visintin, Mei Gong, Cherilyn M Sirois, Brian G Monks & Douglas T Golenbock

  2. Institute of Cancer Research and Molecular Medicine, The Norwegian University of Science and Technology, Trondheim, N-7489, Norway

    Dionne C G Klein & Terje Espevik

  3. Department of Physics, The Norwegian University of Science and Technology, Trondheim, N-7489, Norway

    Dionne C G Klein

  4. Department of Physiology and Biophysics, Boston University School of Medicine, Boston, 02118, Massachusetts, USA

    C James McKnight

  5. Centre for Cancer Research and Cell Biology, School of Biomedical Sciences, The Queen's University of Belfast, Belfast, BT7 9BL, Northern Ireland, United Kingdom

    W Paul Duprex

  6. Eisai Research Institute, Andover, 01810, Massachusetts, USA

    Marc S Lamphier

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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

Correspondence to Eicke Latz or Douglas T Golenbock.

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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)

Supplementary Methods (PDF 152 kb)

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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

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