Structural basis of RNA recognition and activation by innate immune receptor RIG-I - PubMed (original) (raw)

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

Fuguo Jiang et al. Nature. 2011.

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'-ppp and by polyuridine sequences. 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 cancer. 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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Figures

Figure 1

Figure 1. Structural overview of RIG-I helicase-RD

(a, b, and c) Schematic representation of the RIG-I helicase-RD, highlighting the RecA-like domain 1 (blue), the alpha-helical domain 3 (green), RecA-like helicase domain 2 (yellow), and RD (red). The linker connecting Domain 1 with Domain 3 is colored teal, while the V-shape linker between Domain 2 and RD is colored orange. The ADP•BeF3 and dsRNA are shown in stick representation with the 5′ and 3′ strands of the RNA colored black and beige, respectively. A grey sphere denotes the position of the zinc ion in RD. The 3′ and 5′ strands are colored beige and black, respectively. (d, e, f) Surface of RIG-I helicase-RD colored for electrostatic potential at ±5 kT/e; blue (basic), white (neutral), and red (acidic). The views in panels a, b, and c are identical to d, e and f, respectively.

Figure 2

Figure 2. Interactions of RIG-I helicase-RD with dsRNA and ADP•BeF3

A schematic representation showing the interactions between RIG-I domains and helicase motifs (given in parentheses) with dsRNA is located in the center. Detailed contacts are shown in the surrounding panels. Stick representation detailing the RIG-I helicase motifs interactions with ADP•BeF3 and Mg2+ is shown in the lower left panel.

Figure 3

Figure 3. Comparison of RIG-I helicase RD with HCV NS3h and RD bound to 5′-OH and 5′-ppp dsRNA

(a, b, and c) Ribbons diagram showing the superposition of RIG-I helicase-RD•dsRNA•ADP•BeF3 structure and NS3h bound to ssDNA (PDB code 3KQH) (grey). The helicase core domains 1 and 2 from RIG-I helicase-RD superimpose well, while domain 3 of NS3h is positioned over the RD. (b) Superposition of RIG-I helicase-RD with NS3h demonstrates that the ssDNA bound to NS3h overlays with the 3′-strand (beige) of the dsRNA bound to the helicase-RD. (c) The location of the Phe-loop of NS3h relative to the dsRNA of the RIG-I helicase-RD•dsRNA•ADP•BeF3 structure. (d and e) Superposition of the 5′-OH (blue; PDB code 3OG8) and 5′-ppp dsRNA (magenta; PDB code 3LRR) based on the location of RD. For clarity the 5′ strands (d) and 3′ strands (e) are shown separately.

Figure 4

Figure 4. Limited trypsin digestion, DSF and SAXS analyses of helicase-RD and full-length RIG-I in the presence and absence of dsRNA

(a) SDS-PAGE analysis of a time course (minutes) of limited trypsin digestion of helicase-RD or full-length RIG-I in the absence or presence of 14 base pair pal-dsRNA. (b) DSF of RIG-I helicase-RD or full-length RIG-I in the presence of 14 base pair pal-dsRNA and/or ADP•BeF3 with respect to protein alone. The bar graph displays the mean melting temperature difference (Δ_T_m) and the error bars represent the standard deviation from three independent measurements. (c and d) Ab initio envelope of helicase-RD and dsRNA overlaid with the crystal structure of helicase-RD•dsRNA (dsRNA truncated to 10 base pairs). The view in d is rotated 90° about a horizontal axis from panel c. (e and f) Ab initio envelope of full-length RIG-I and dsRNA overlaid with the crystal structure of helicase-RD•dsRNA with two copies of CARDs added (PDB code 2VGQ). The view in f is rotated 90° about a horizontal axis from panel e.

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