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Structural basis of RNA recognition and activation by innate immune receptor RIG-I

Nature volume 479, pages 423–427 (2011)Cite this article

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Abstract

Retinoic-acid-inducible gene-I (RIG-I; also known as DDX58) is a cytoplasmic pathogen recognition receptor that recognizes pathogen-associated molecular pattern (PAMP) motifs to differentiate between viral and cellular RNAs. RIG-I is activated by blunt-ended double-stranded (ds)RNA with or without a 5′-triphosphate (ppp), by single-stranded RNA marked by a 5′-ppp1 and by polyuridine sequences2,3. Upon binding to such PAMP motifs, RIG-I initiates a signalling cascade that induces innate immune defences and inflammatory cytokines to establish an antiviral state. The RIG-I pathway is highly regulated and aberrant signalling leads to apoptosis, altered cell differentiation, inflammation, autoimmune diseases and cancer4,5. The helicase and repressor domains (RD) of RIG-I recognize dsRNA and 5′-ppp RNA to activate the two amino-terminal caspase recruitment domains (CARDs) for signalling. Here, to understand the synergy between the helicase and the RD for RNA binding, and the contribution of ATP hydrolysis to RIG-I activation, we determined the structure of human RIG-I helicase-RD in complex with dsRNA and an ATP analogue. The helicase-RD organizes into a ring around dsRNA, capping one end, while contacting both strands using previously uncharacterized motifs to recognize dsRNA. Small-angle X-ray scattering, limited proteolysis and differential scanning fluorimetry indicate that RIG-I is in an extended and flexible conformation that compacts upon binding RNA. These results provide a detailed view of the role of helicase in dsRNA recognition, the synergy between the RD and the helicase for RNA binding and the organization of full-length RIG-I bound to dsRNA, and provide evidence of a conformational change upon RNA binding. The RIG-I helicase-RD structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA. The structure yields unprecedented insight into innate immunity and has a broader impact on other areas of biology, including RNA interference and DNA repair, which utilize homologous helicase domains within DICER and FANCM.

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Figure 1: Structural overview of RIG-I helicase-RD.
Figure 2: Interactions of the RIG-I helicase-RD with dsRNA and ADP•BeF3.
Figure 3: Comparison of RIG-I helicase-RD with HCV NS3h and RD bound to 5′-OH and 5′-ppp dsRNA.
Figure 4: Limited trypsin digestion, DSF and SAXS analyses of helicase-RD and full-length RIG-I in the presence and absence of dsRNA.

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Protein Data Bank

Data deposits

Atomic coordinates have been deposited in the Protein Data Bank under accession code 3TMI.

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Acknowledgements

We acknowledge access to beamlines X29 at the NSLS (National Synchrotron Light Source), LRL-CAT at APS (Advanced Photon Source), and G1 and F1 at CHESS (Cornell High Energy Synchrotron Source) and thank the NSLS, APS and CHESS staff. NSLS and APS are supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886 and DE-AC02-06CH11357, respectively. CHESS is supported by the NSF and NIH/NIGMS through NSF award DMR-0936384, and the MacCHESS resource is supported by NIH/NCRR award RR-01646. Use of the LRL-CAT beamline facilities at Sector 31 was provided by Eli Lilly & Company. We would like to thank V. Rajagopal for initiating the biochemical experiments and guiding the project in the early stages. We thank E. Arnold, H. Berman, S. K. Burley, R. Gillilian, L. Morisco, W. Olson, T. Saito, A. Shatkin, A. Stock, H. Yang and M. Zhuravieva for providing helpful comments and assistance. This work was supported by NIH grants GM55310 to S.S.P. and AI080659 to J.M.

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Author notes

  1. Fuguo Jiang and Anand Ramanathan: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Center for Advanced Biotechnology and Medicine, Rutgers University, 679 Hoes Lane West, Piscataway, New Jersey 08854, USA,

    Fuguo Jiang, Matthew T. Miller & Joseph Marcotrigiano

  2. Department of Biochemistry, UMDNJ-RWJ Medical School, 675 Hoes Lane West, Piscataway, New Jersey 08854, USA,

    Anand Ramanathan, Guo-Qing Tang & Smita S. Patel

  3. Department of Immunology, University of Washington School of Medicine, 1959 NE Pacific Street, Seattle, Washington 98195, USA,

    Michael Gale

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Contributions

The project was initiated by M.G., J.M. and S.S.P. J.M. and S.S.P. designed and supervised the project. M.G. provided reagents and consultation. F.J. designed protein constructs and established purification protocols. A.R. generated all RNA reagents. F.J. and A.R. purified the complex and set up crystallization screens. F.J. optimized the crystal for data collection. J.M., M.T.M., F.J. and A.R. collected, processed and analysed the X-ray crystallographic data. M.T.M., F.J. and J.M. collected and analysed the SAXS data. A.R., G.-Q.T. and S.S.P. collected and analysed the RNA binding and ATPase assays. F.J. performed limited proteolysis and thermal melting assay. S.S.P. and J.M. wrote the paper and all authors contributed to editing.

Corresponding authors

Correspondence to Smita S. Patel or Joseph Marcotrigiano.

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The authors declare no competing financial interests.

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Jiang, F., Ramanathan, A., Miller, M. et al. Structural basis of RNA recognition and activation by innate immune receptor RIG-I. Nature 479, 423–427 (2011). https://doi.org/10.1038/nature10537

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