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

  • ️Sat Jan 01 2011

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 (ΔTm) 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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