Assembly of the RIG-I 2CARD Signaling Complex

Abstract

The innate immune system of mammals utilizes pattern-recognition receptors to differentiate between pathogen associated molecular patterns and host molecules. The retinoic acid inducible-gene-I (RIG-I) like receptors (RLRs) bind viral dsRNAs, triggering an innate immune response. RLRs contain two N-terminal caspase activation and recruitment domains (2CARD), a DExD/H-box helicase-like domain, and a C-terminal regulatory domain. In the absence of RNA, the 2CARD region is sequestered by the helicase domains in an autoinhibited state. Upon binding to the 5′-ends of activating viral dsRNAs a conformational change exposes 2CARD. The CARD domains then interact with MAVS, a mitochondrial outer membrane protein, leading to downstream signaling and interferon expression. Signaling is modulated by K63-linked polyubiquitin chains (polyUb). It has been reported that polyUb binding to 2CARD induces formation of 2CARD tetramer. However, the nature of the assembly process is not well understood and we are investigating the thermodynamic linkage between polyUb binding and 2CARD oligomerization using sedimentation velocity analytical ultracentrifugation. 2CARD alone exists as a monomer; however, in the presence of polyUb several high molecular weight complexes are formed. The assembly process is dependent on salt, polyUb concentration, and polyUb chain length.