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Toll-like receptor 8 senses degradation products of single-stranded RNA

Abstract

Toll-like receptor 8 (TLR8) recognizes viral or bacterial single-stranded RNA (ssRNA) and activates innate immune systems. TLR8 is activated by uridine- and guanosine-rich ssRNA as well as by certain synthetic chemicals; however, the molecular basis for ssRNA recognition has remained unknown. In this study, to elucidate the recognition mechanism of ssRNA, we determined the crystal structures of human TLR8 in complex with ssRNA. TLR8 recognized two degradation products of ssRNA—uridine and a short oligonucleotide—at two distinct sites: uridine bound the site on the dimerization interface where small chemical ligands are recognized, whereas short oligonucleotides bound a newly identified site on the concave surface of the TLR8 horseshoe structure. Site-directed mutagenesis revealed that both binding sites were essential for activation of TLR8 by ssRNA. These results demonstrate that TLR8 is a sensor for both uridine and a short oligonucleotide derived from RNA.

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Figure 1: Structures of human TLR8.
Figure 2: ssRNA recognition by TLR8.
Figure 3: LC-MS analyses of dissolved TLR8–ORN06 crystals.
Figure 4: Ligand recognition at the first site of TLR8.
Figure 5: Binding affinities for mononucleosides and a synergistic effect of uridine and oligonucleotide.
Figure 6: Proposed mechanism of TLR8 activation by binding of ssRNA.

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Acknowledgements

We thank the beamline staff members at Photon Factory and SPring-8 for their assistance with data collection. We also thank M. Osawa and I. Shimada for their help with the ITC data acquisition. This work was supported by Grants-in-Aid from the Japan Society for the Promotion of Science KAKENHI, grant nos. 25121709, 25115504, 26711002 (U.O.), 25860354 (T. Shibata), 25253032 (K.M.), 23116007 and 25291010 (T. Shimizu); by the Takeda Science Foundation (U.O. and T. Shimizu); and by the Mochida Memorial Foundation for Medical and Pharmaceutical Research (U.O.).

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H.T. and U.O. performed the crystallization and structure determination. T. Shibata and K.M. performed the functional analyses. M.T., Y.Y. and T.I. performed LC-MS. H.T., U.O., T. Shibata, K.M. and T. Shimizu analyzed the results. All authors contributed to writing the manuscript.

Corresponding author

Correspondence to Toshiyuki Shimizu.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Structures of TLR8 in complex with ssRNA.

(a, b) Structures of TLR8–ssRNA40 (a) and TLR8–ORN06S (b). Front (left) and side (right) views of the structures. Uridine is recognized at the 1st site, and UG is recognized at the 2nd site.(c) Surface representations of a protomer of TLR8–ORN06 dimer. The protein-protein interface is shown in orange, the 1st site is shown in red, and the 2nd site is shown in purple.(d) Superposition of oligonucleotides bound at the 2nd sites of TLR8–ORN06, TLR8–ssRNA40 and TLR8–ORN06S. UG in TLR8–ORN06, TLR8–ssRNA40 and TLR8–ORN06S are shown in purple, cyan and orange, respectively.

Supplementary Figure 2 Electron density maps for the bound ligand.

(a-d) Electron density (shown in gray) contoured around the ligand at the 3.0σ level in the Fo-Fc map; uridine and UG in TLR8– ORN06 (a), TLR8–ssRNA40 (b), TLR8– ORN06S (c) and uridine in TLR8–uridine (d).

Supplementary Figure 3 Close-up views of the first and second sites of TLR8–ssRNA40 and TLR8–ORN06S.

(a) The 1st site of TLR8–ssRNA40. Uridine (shown in yellow) was modeled into the structure.(b) The 2nd site of TLR8–ssRNA40. Electron density corresponding to a pyrimidine-purine dinucleotide was observed at the 2nd site and assigned tentatively as UG (shown in purple).(c) The 1st site of TLR8–ORN06S. Uridine was modeled into the structure, and is shown in yellow.(d) The 2nd site of TLR8–ORN06S. UG was modeled into the structure, and is shown in purple.

Supplementary Figure 4 Sequence alignment of human, bovine and mouse TLR7 and TLR8.

Sequence alignments are displayed for each LRR module. The 1st and 2nd sites deduced from chain A of the TLR8–ORN06 complex are indicated by yellow and blue highlightings, respectively. The residues missing in the structure are highlighted in grey. Alignments were performed using CLUSTALW software (EMBL-European Bioinformatics Institute). Residues are colored to indicate the degree of similarity; red residues display the highest similarity, followed by green, blue, and then black (lowest similarity).

Supplementary Figure 5 Structures of TLR8 in complex with uridine.

(a) Front (left) and side (right) views of the structure. Uridine is recognized at the 1st site.(b) Residues involved the interaction of TLR8 with uridine. The C atoms of the ligand molecules are shown in yellow. Water molecules mediating ligand recognition are indicated by red spheres, and hydrogen bonds are indicated by dashed lines.

Supplementary Figure 6 Small-angle X-ray scattering analysis of TLR8 in complex with ssRNA.

Scattering curves of hTLR8 by SAXS experiments are shown. The proteins used for SAXS were unliganded hTLR8 (red), hTLR8 in the presence of ORN06 (blue), and hTLR8 in the presence of R848 (black).

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Tanji, H., Ohto, U., Shibata, T. et al. Toll-like receptor 8 senses degradation products of single-stranded RNA. Nat Struct Mol Biol 22, 109–115 (2015). https://doi.org/10.1038/nsmb.2943

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