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. 2016 May 19;62(4):586-602.
doi: 10.1016/j.molcel.2016.04.021.

Structural Analysis of dsRNA Binding to Anti-viral Pattern Recognition Receptors LGP2 and MDA5

Emiko Uchikawa  1 , Mathilde Lethier  1 , Hélène Malet  1 , Joanna Brunel  2 , Denis Gerlier  2 , Stephen Cusack  3
Affiliations

Structural Analysis of dsRNA Binding to Anti-viral Pattern Recognition Receptors LGP2 and MDA5

Emiko Uchikawa et al. Mol Cell. .

Abstract

RIG-I and MDA5 sense virus-derived short 5'ppp blunt-ended or long dsRNA, respectively, causing interferon production. Non-signaling LGP2 appears to positively and negatively regulate MDA5 and RIG-I signaling, respectively. Co-crystal structures of chicken (ch) LGP2 with dsRNA display a fully or semi-closed conformation depending on the presence or absence of nucleotide. LGP2 caps blunt, 3' or 5' overhang dsRNA ends with 1 bp longer overall footprint than RIG-I. Structures of 1:1 and 2:1 complexes of chMDA5 with short dsRNA reveal head-to-head packing rather than the polar head-to-tail orientation described for long filaments. chLGP2 and chMDA5 make filaments with a similar axial repeat, although less co-operatively for chLGP2. Overall, LGP2 resembles a chimera combining a MDA5-like helicase domain and RIG-I like CTD supporting both stem and end binding. Functionally, RNA binding is required for LGP2-mediated enhancement of MDA5 activation. We propose that LGP2 end-binding may promote nucleation of MDA5 oligomerization on dsRNA.

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Figures

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Graphical abstract
Figure 1
Figure 1
Overall Structure of chLGP2-dsRNA-ADP:AlF4 Complex (A) Domain structure of chLGP2. Domain colors are green (Hel1), yellow (Hel2i), cyan (Hel2), red (pincer motif), and orange (CTD). (B) Side and head-end cartoon view of chLGP2-dsRNA-ADP:AlF4 complex (left and middle) compared with hRIG-I-dsRNA-ADP:BeF3 complex (PDB: 5E3H [3TMI]) (right). In the side view, the head end contains the pincer motif and the tail end contains the CTD. Domain colors are as in (A), with the zinc atom in the CTD a black sphere. The dsRNA 3′ and 5′ strands are, respectively, violet and yellow. The ADP:AlF4 is in spheres representation. Note that the second pincer domain helix (α19) of chLGP2 extends right up to the CTD, unlike in hRIG-I. (C) Details of the immediate protein ligands of the ADP:AlF4:Mg2+ bound in chLGP2, all of which (except Glu67) come from helicase motifs Q, I and II (Hel1), and Va and VI (Hel2), which are colored as indicated. The Mg2+ ion, aluminum, and fluorine atoms are, respectively, purple, gray, and light blue spheres. Glu132 and Gln465 coordinate the mimic of the attacking water molecule (red sphere) in this transition-state analog complex. (D) Diagram showing how in chLGP2, Glu67 emerges from helix a3 to be involved in ATP ribose binding and stabilization of Arg32, which stacks on the adenine base (top). A very similar situation occurs in MDA5 (see Figure 4D) but not RIG-I (bottom). (E) Left: table of Km and kcat values for the RNA-dependent ATPase activity of wild-type chLGP2, dRIG-I, and chMDA5. Right: representative ATP hydrolysis curves for chLGP2 and hLGP2 with without dsRNA and various mutations of chLGP2. See also Figure S2. See also Figures S1–S3.
Figure 2
Figure 2
dsRNA Binding by chLGP2 (A) Surface representation of the chGP2-dsRNA-ADP:AlF4 complex viewed from the tail (left) and head (right) ends, colored as in Figure 1. At the tail end, the protein completely caps the blunt end of the dsRNA, whereas at the head end, the dsRNA can be extended. (B) Schematic diagram showing the interactions of chLGP2 residues with the 10-mer dsRNA. Residues are colored according to domain and labeled with the conserved motif they belong to and the atom involved in the interaction. Polar interactions are indicated with a blue dotted line (cutoff 3.5 Å) and hydrophobic interactions by an arc. Numerous direct or water-mediated interactions are omitted for clarity. The 3′ strand nucleotides are numbered with an asterisk (i.e., 5′-G1:C1∗-3′ is the first base pair from the tail end). The two observed alternative conformations of the first and second 5′ phosphates are shown. (C) Details of the chLGP2-RNA interactions that cap the blunt end of the 5′ppp dsRNA showing the role of aromatic residues Phe595, Phe599, and Trp602 from the capping loop, Glu571 and His574 from the 3′ end-binding loop, and His406 from the Hel2-loop. The Mg2+ coordinated by the 5′ppp is a magenta sphere. (D) Comparison of chLGP2 structures with 5′ppp-dsRNA without (gray) or with (colors) a 3′-GG overhang, showing how a slight displacement of the capping loop and rearrangement of the β3-α4 loop accommodates the 3′ end extrusion. (E) Schematic diagram showing interactions with the 3′-GG overhang nucleotides (denoted G-1∗ and G-2∗), annotated as in (B). (F) Structural details of the network of interactions between chLGP2 and the 3′-GG overhang nucleotides. Compared with (C), Glu571 and His574 no longer interact with C1∗ ribose. See also Figures S4 and S5.
Figure 3
Figure 3
Comparison of chLGP2 and hRIG-I Binding to dsRNA (A) Structural and schematic diagrams comparing the mode of end binding of chLGP2 and hRIG-I illustrating the extra base pair sequestered by chLGP2, which is at the same level as Phe853 from the capping loop of RIG-I. Conserved interactions with motifs Ia, Ib, and Ic are shown. (B) Kd values between chLGP2 (green), full-length hRIG-I (red), and chMDA5 (blue) and a 12-mer dsRNA with different 5′ modifications were measured without nucleotide (no NTP) and with various nucleotides (ADP-AlF4, ADP, ATP). The values shown correspond to Kd (nM) ± SD on the basis of the fluorescence anisotropy binding curves shown in Figure S5B. See also Figures S4 and S5.
Figure 4
Figure 4
Overall Structure of chMDA5ΔCARD-ADP Complex (A) Domain structure of chMDA5ΔCARD, omitting the N-terminal tandem CARD domains. (B) Ribbon diagram of the chMDA5ΔCARD-10-mer dsRNA-ADP complex from head end along (left) and perpendicular (right) to the dsRNA axis, colored according to Figure 2A. (C) Diagram showing the interactions of chMDA5 with ADP:Mg. (D) Diagram showing how Glu369 emerges from helix a3 to be involved in ribose binding and stabilization of Arg330, which stacks on the adenine base. A very similar situation occurs in LGP2 but not RIG-I (see Figure 1D). (E) Ribbon diagram of the head-to-head chMDA5ΔCARD-24-mer dsRNA-ADP complex perpendicular to the dsRNA axis. Domains and dsRNA are colored according to Figure 2A, with lighter colors for the second monomer.
Figure 5
Figure 5
Interactions of chMDA5 with dsRNA (A) Head and tail views down the dsRNA axis of the chMDA5 structure showing that the dsRNA can continue from both ends (see also D). Compared with LGP2 (Figure 2A), MDA5 appears from the tail end as an open horseshoe rather than a disk. (B) Schematic diagram showing the interactions of chMDA5 residues with the 10-mer dsRNA, annotated as in Figure 2B. (C) Ribbon diagram showing protein-RNA interactions in the chMDA5 10-mer structure at different levels along the dsRNA, including those from helix α12 of Hel2i, the Hel2 loop, and two loops of the CTD. The putative position of the disordered MDA5 capping loop is shown dotted on the basis of the crystal structure of the isolated hMDA5 CTD. Protein-RNA interactions mediated by the Hel1 and Hel2 domains have been omitted for clarity (see B). (D) As in (C), but the dsRNA has been extended by 3 bp to emerge from the tail end of the molecule. Modeling suggests that the MDA5 capping loop could be involved in another level of protein-RNA interactions as well as possibly mediating the tail-to-head protein-protein interface.
Figure 6
Figure 6
Dimer and Filament Formation by chMDA5 and chLGP2 (A) Comparison of monomeric and dimeric structures of MDA5-dsRNA complexes. hMDA5 12-mer (PDB: 4GL2) (left), chMDA5 10-mer (middle-left), chMDA5 24-mer (middle-right), and chMDA5 27-mer (right). In all cases, the bottom molecule (green ribbons with CTD in orange, pincer domain in red) is in the same orientation. The top molecule is cyan except for the pincer domain (pink). The chMDA5 10-mer and 24-mer structures are essentially the same apart from the lack of continuity of the dsRNA in the former. In both cases, the axis of the dsRNA is bent by 35° between the two head-to-head molecules, but in the chMDA5 27-mer complex, it is straight. (B) Schematic representation of (A) highlighting the dsRNA conformation. The distance between the center of mass of the two molecules in the head-to-head dimer is indicated. Compared with the hMDA5 12-mer structure (left), the chMDA5 10-mer structure (middle left) lacks 3 bp at the tail end and gains 1 bp at the head end, and there is a 1 bp gap between the RNAs in the two head-to-head molecules in the dimer (dotted base pairs indicate potential extra base pairs not in the structure). In the chMDA5 24-mer (middle right), the dsRNA is continuous between the molecules in the dimer but bent. Because there is 1 bp between the two MDA5 molecules and 24 bp overall, the structure is likely a superposition of structures with either 11 or 12 bp bound to one MDA5 (i.e., 11.5 on average), as indicated by the dashed line. In the low-resolution chMDA5 27-mer, it is unclear whether there are extra base pairs between the molecules, which are more widely separated, or at the tail ends, but the dsRNA is straight. (C) Detail of the four regions involved in protein-protein interactions mediating the head-to-head packing. (D) EM analysis of chLGP2:Φ6 dsRNA complexes in the presence of ATP. Left: molar ratio 0.2:1 with uranyl acetate negative stain. Middle left: molar ratio 1:1 with negative stain. Middle right: molar ratio 1:1 with cryo-EM. Right: cryo-EM image 2D class average and power spectrum. (E) EM analysis of chMDA5:Φ6 dsRNA complexes in the presence of ATP. Left: molar ratio 0.2:1 with negative stain. Middle left: molar ratio 1:1 with negative stain. Middle right: molar ratio 1:1 with cryo-EM. Right: cryo-EM image 2D class average and power spectrum.
Figure 7
Figure 7
Cooperativity of LGP2 with MDA5 in Cells (A) Enhancement of poly(I:C)-mediated activation of endogenous chMDA5 by exogenously supplied chLGP2 (27.5 ng DNA/well) in chicken DF1 cells. See also Figures S7A–S7C. (B) Effect of chLGP2, chLGP2 variants (K138E/R490E or hel°, K648E/K649E or CTD°, K138E/R490E/K648E/K649E or hel°CTD°), and hLGP2 (27.5 ng DNA/well) on the activation of chIFNβ promoter by endogenous chMDA5 stimulated (yellow bars) or not (blue bars) by poly(I:C) and by exogenous chMDA5 (1.67 ng DNA /well) in the absence (black bars) and presence (green bars) of poly(I:C) in chicken DF1 cells. ∗p < 0.05 and ∗∗p > 0.025 and below. Comparison of endogenous MDA5 and exogenous MDA5 co-transfected or not with LGP2 and activated or not with poly(I:C) are in black, LGP2 activated chLGP2 variants with WT counterpart are in red (effect on endogenous chMDA5), blue (effect on exogenous chMDA5), and green (effect on exogenous chMDA5 + poly[I:C]). Data are mean ± SD of three independent experiments, with each combination done in triplicate each time. See also Figures S7D–S7F. (C) Effect of hLGP2 on the activation of exogenous hMDA5 and hMDA5 IE/KR (1.67 ng DNA/well) (I841K/E842R) mutant by poly(I:C) (denoted [I:C]) in the absence (white bars) and presence (black bars) of exogenously supplied hLGP2 (27.5 ng DNA/well) in human Huh7.5 cells. ∗∗p < 0.01 and ∗∗∗p > 0.0025. Background signal (mean value) of cells transfected with control plasmid DNA followed or not by transfection of poly(I:C) is indicated by the shaded area. See also Figure S7G. See also Figure S7.
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