Figure 2. The RIG-I Singleton-Merten syndrome variant C268F is catalytically dead and has reduced ATP-binding-properties.

(A) ATP hydrolysis activity of RIG-I, the RIG-I Singleton-Merten syndrome (SMS) variant C268F and the RIG-I motif I and II mutants K270I and E373Q. RIG-I proteins were incubated with [γ-³²P]-ATP in the presence or absence of a 12mer dsRNA for 15 min at room temperature and free phosphate was separated from ATP by thin layer chromatography. (B) Affinity of RIG-I, RIG-I C268F and the RIG-I motif I and II mutants to MANT-ATP or MANT-ATPγS measured by tryptophan fluorescence Förster resonance energy transfer to the MANT-nucleotide. Proteins were incubated with increasing amounts of nucleotides in the presence or absence of a 14mer dsRNA. MANT fluorescence was recorded minus a MANT-nucleotide-only control. n = 4, error bars represent mean values ± SD. (C) 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 2—figure supplement 1. Location of RIG-I amino-acid substitutions used in Figure 2.

A RIG-I-RNA-ADP·BeF₃ structure (PDB: 5E3H) served as scaffold (Jiang et al., 2011). The RIG-I SF2 sub-domains (1A, 2A and 2B) are colored in light blue and dark blue. The RD is depicted in cyan and 2CARD is indicated in yellow. Mutated amino acid side chains are depicted in orange. Q247 and R244 are located within the SF2 Q-motif and participate in ATP base recognition. C268 is mutated in atypical Singleton-Merten syndrome.