RIG-I-like receptors (RLRs), RIG-I, MDA5, and LGP2, are a family of innate immune receptors that recognize viral RNA in the cytoplasm and initiate antiviral responses including the induction of type I interferons and other pro-inflammatory cytokines. All three proteins have both an RNA helicase domain with ATPase activity and a C-terminal domain (CTD) which is responsible for RNA binding. RIG-I and MDA5 also have two tandem caspase activation and recruitment domains (CARDs) at the N-terminus which are involved in downstream signaling. To understand the structural basis of viral RNA recognition by the RLRs, especially RIG-I, we have performed extensive biochemical studies to determine the binding properties of RIG-I with different forms of RNA, including dsRNA with and without 5'-triphosphate (5'-ppp) groups, and 5'-ppp ssRNA. RIG-I CTD binds to these forms of RNA, and exhibits the highest affinity for 5'-ppp dsRNA. We also determined the crystal structures of RIG-I CTD in complex with dsRNA with and without 5'-ppp by X-ray crystallography. The structures showed that RIG-I recognizes the termini of the dsRNA and interacts with the two types of RNA in different orientations. By comparing these complex structures together with mutagenesis studies, we conclude that RIG-I CTD is a versatile binding module capable of recognizing different RNA ligands. Similar but partially differing sets of residues are involved in the recognition of dsRNA with and without 5'-ppp. Mutations of key residues at the RNA binding surface also abolished RIG-I signaling in cells. In order to compare the RIG-I/RNA interactions with other RLRs, we also determined the dsRNA binding surface of MDA5 CTD by NMR titration studies. MDA5 CTD has a similar binding surface to that of RIG-I CTD, however with slightly different surface electrostatic potentials which indicate different interactions with RNA. This may explain how MDA5 senses differing types of viruses compared to RIG-I. The current RIG-I activation model suggests that after stimulation by RNA binding, RIG-I undergoes an ATP-dependent conformational change, exposing the CARDs for downstream signaling. To understand the critical role that the helicase domain plays in RIG-I activation by structural approach, we also attempted to crystallize the dsRNA-bound helicase domain together with CTD.
