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. 2011 Mar;39(4):1565-75.
doi: 10.1093/nar/gkq974. Epub 2010 Oct 20.

Crystal structure of RIG-I C-terminal domain bound to blunt-ended double-strand RNA without 5' triphosphate

Cheng Lu  1 , C T Ranjith-Kumar, Lujiang Hao, C Cheng Kao, Pingwei Li
Affiliations

Crystal structure of RIG-I C-terminal domain bound to blunt-ended double-strand RNA without 5' triphosphate

Cheng Lu et al. Nucleic Acids Res. 2011 Mar.

Abstract

RIG-I recognizes molecular patterns in viral RNA to regulate the induction of type I interferons. The C-terminal domain (CTD) of RIG-I exhibits high affinity for 5' triphosphate (ppp) dsRNA as well as blunt-ended dsRNA. Structures of RIG-I CTD bound to 5'-ppp dsRNA showed that RIG-I recognizes the termini of dsRNA and interacts with the ppp through electrostatic interactions. However, the structural basis for the recognition of non-phosphorylated dsRNA by RIG-I is not fully understood. Here, we show that RIG-I CTD binds blunt-ended dsRNA in a different orientation compared to 5' ppp dsRNA and interacts with both strands of the dsRNA. Overlapping sets of residues are involved in the recognition of blunt-ended dsRNA and 5' ppp dsRNA. Mutations at the RNA-binding surface affect RNA binding and signaling by RIG-I. These results provide the mechanistic basis for how RIG-I recognizes different RNA ligands.

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Figures

Figure 1.
Figure 1.
RIG-I CTD binds blunt-ended dsRNA without 5′ ppp. (A) Binding studies of RIG-I CTD with the 14-bp blunt-ended dsRNA by gel filtration chromatography. RIG-I CTD, the 14-bp blunt-ended dsRNA and mixtures of RIG-I CTD:dsRNA at molar ratios 1:1 to 4:1 were injected over a superdex200 column. Elution profile of RIG-I CTD is in black and the dsRNA in red. Elution profiles of mixtures of RIG-I CTD and the 14-bp dsRNA at molar ratios 1:1, 2:1, 3:1 and 4:1 are in green, blue, cyan and purple, respectively. The molecular masses of four protein standards and their elution positions are shown above the chromatograms. (B) Binding studies of RIG-I CTD with a 14-bp dsRNA containing 5′ AUAU overhangs. A 2:1 mixture of RIG-I CTD and the dsRNA was analyzed by gel filtration chromatography. (C) Binding studies of RIG-I CTD with a 14-bp dsRNA containing 3′AUAU overhangs. The molar ratio between RIG-I CTD and the dsRNA is 2:1 in the sample. (D) Binding studies of RIG-I CTD with a 14-bp blunt-ended dsDNA with the same sequence as the 14-bp blunt-ended dsRNA. The molar ratio between RIG-I CTD and the dsDNA is 2:1. (E) Binding studies of RIG-I CTD with a 13-bp RNA:DNA hybrid. The molar ratio between RIG-I CTD and the RNA:DNA hybrid is 2:1. (F) Binding studies of RIG-I CTD with the 14-bp 5′ ppp dsRNA, 14-bp blunt-ended dsRNA and 14-bp dsRNA with either 5′ or 3′ overhangs by EMSA.
Figure 2.
Figure 2.
Crystal structure of human RIG-I CTD bound to a 14-bp blunt-ended dsRNA. (A) Structure of RIG-I CTD bound to the dsRNA. RIG-I CTDs are shown by the green ribbons. The dsRNA is shown by the sticks representation. Carbon atoms of the two RNA strands are colored cyan and pink, respectively. The zinc ion bound to RIG-I CTD is shown by the gray sphere. The complex exhibits pseudo 2-fold non-crystallographic symmetry. The pseudo 2-fold axis is shown by the black oval. (B) Surface electrostatics of RIG-I CTD. Positively charged surface is colored blue and negatively charged surface red. The blunt-ended dsRNA bound to RIG-I CTD is shown by the stick models. Key residues mediating blunt-ended dsRNA recognition are labeled. Location of the ppp-binding site for 5′ ppp dsRNA is indicated by the white asterisk.
Figure 3.
Figure 3.
Structural basis of blunt-ended dsRNA recognition by RIG-I CTD. (A) Interactions between the 5′ 4 nt of the 14-bp blunt-ended dsRNA and RIG-I CTD. The dsRNA is shown by the stick models. Carbon atoms of the RNA strand interacting with RIG-I CTD are colored cyan. The complementary strand is colored light gray. RIG-I CTD is shown by the green ribbons. Key residues involved in RNA binding are shown by the magenta stick models. (B) Interactions between complementary strand and RIG-I CTD. The complementary strand is shown by the stick models with carbon atoms colored pink. (C) Schematic representations of the interactions between RIG-I CTD and the 14-bp blunt-ended dsRNA (left) and a 14-bp 5′ ppp dsRNA (right). Nucleotides in the two dsRNA are labeled the same way for comparison. Interactions between the dsRNA and one of the two RIG-I CTD molecules are shown.
Figure 4.
Figure 4.
Mutations at the blunt-ended dsRNA-binding surface affect RNA binding by RIG-I CTD. (A) Binding studies of wild-type and mutants of RIG-I CTD with the 14-bp blunt-ended dsRNA by EMSA. (B) Binding studies of RIG-I CTD mutants with a 14-bp 5′ ppp dsRNA. (C) Binding studies of RIG-I CTD mutants with a 13-nt 5′ ppp ssRNA.
Figure 5.
Figure 5.
RIG-I and LGP2 CTDs bind blunt-ended dsRNA and 5′ ppp dsRNA differently. (A) Superposition of RIG-I CTD bound to the 14-bp blunt-ended dsRNA and a 14-bp 5′ ppp dsRNA with the same sequence. RIG-I CTDs bound to the blunt-ended dsRNA (blue) and the 5′ ppp dsRNA (red) are shown by the green and cyan ribbons, respectively. (B) Superposition of RIG-CTD bound to the blunt-ended dsRNA and the 5′ ppp dsRNA and LGP2 CTD bound to blunt-ended dsRNA. The blunt-ended dsRNA bound to RIG-I CTD is shown by the blue ribbons. The 5′ ppp dsRNA bound to RIG-I CTD is shown by the orange ribbons. A 14-bp dsRNA (magenta ribbons) is superimposed on the 8-bp dsRNA in the LGP2 CTD:dsRNA complex structure to facilitate comparisons. (C) Superposition of RIG-I (ligand bound), MDA5 (ligand free) and LGP2 (ligand bound) CTD structures. The RNA-binding surfaces of the proteins face the reader. (D) Electrostatics of the RNA-binding surfaces of the RLR CTDs. Positively charged surfaces are colored blue and negatively charged surfaces are red. The ppp-binding site of RIG-I CTD and corresponding regions of LGP2 and MDA5 are indicated by the green circles. Key residues involved in RNA-binding are labeled. Residues of RIG-I CTD interacting with the complementary strand of the dsRNA and corresponding residues in MDA5 and LGP2 CTDs are underlined.
Figure 6.
Figure 6.
Mutations at the RNA-binding surface affect RIG-I signaling. (A) Effects of structures of the RNA ligands on the activation of IFN-β luciferase production. The assay shows the activities of a 27-nt 5′ ppp ssRNA (3PssR27), a 24-bp 5′ ppp dsRNA (3PdsR24) and a 27-bp blunt-ended dsRNA (dsR27) in stimulating RIG-I signaling in HEK 293T cells. The error bars correspond to the standard deviations of signals from three independent transfections. (B) Effects of mutations at the RNA-binding surface on IFN-β luciferase production in cells transfected with a 27-bp blunt-ended dsRNA and a 24-bp dsRNA with 5′ ppp. Expression of the wild-type and mutants of RIG-I in the transfected cells were confirmed by western blot (inset).
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