Figure 3. The RIG-I Singleton-Merten syndrome variant C268F induces amino acid side chain rearrangements within the active site that interfere with nucleotide binding.

(A) ATP-binding pockets of the RIG-I Singleton-Merten syndrome (SMS) variant C268F (left and middle panels) and the RIG-I wild type (right panel) bound to a 14mer dsRNA. The RIG-I SF2 sub-domains are colored in light gray or light blue (1A and 2A) and dark blue (2B). The RD is depicted in cyan and 2CARD is indicated in yellow. (B) Fold change of interferon (IFN)-β promoter-driven luciferase activity in uninfected HEK293T RIG-I KO cells or in cells stimulated with a 19mer 5’-triphosphate (ppp)-dsRNA upon overexpression of different RIG-I mutants. Cells were co-transfected with RIG-I expression vectors and p-125luc/pGL4.74 reporter plasmids, and stimulated with ppp-dsRNA 6 hr post transfection. Firefly luciferase activities were determined in respect to Renilla luciferase activities 16 hr after RNA stimulation. All ratios were normalized to an empty vector control. n = 4–12, error bars represent mean values + SEM.

Figure 3—figure supplement 1. Structural comparison of the RIG-I Singleton-Merten syndrome variant C268F with wild type RIG-I.

(A) Alignment of RIG-I Δ2CARD C268F (light gray) with wild type RIG-I Δ2CARD (color code as in Figure 1—figure supplement 1) PDB 5E3H (Jiang et al., 2011). ADP·Bef₃ and Mg²⁺ are co-crystalized with wild type RIG-I Δ2CARD but not with RIG-I Δ2CARD C268F. (B) 2F_o − F_c electron density of residues within the ATP-binding pocket of RIG-I Δ2CARD C268F at a contour level of 1σ.