Oligomerization of RIG‐I and MDA5 2CARD domains

The innate immune system is the first line of defense against invading pathogens. The retinoic acid‐inducible gene I (RIG‐I) like receptors (RLRs), RIG‐I and melanoma differentiation‐associated protein 5 (MDA5), are critical for host recognition of viral RNAs. These receptors contain a pair of N‐terminal tandem caspase activation and recruitment domains (2CARD), an SF2 helicase core domain, and a C‐terminal regulatory domain. Upon RLR activation, 2CARD associates with the CARD domain of MAVS, leading to the oligomerization of MAVS, downstream signaling and interferon induction. Unanchored K63‐linked polyubiquitin chains (polyUb) interacts with the 2CARD domain, and in the case of RIG‐I, induce tetramer formation. However, the nature of the MDA5 2CARD signaling complex is not known. We have used sedimentation velocity analytical ultracentrifugation to compare MDA5 2CARD and RIG‐I 2CARD binding to polyUb and to characterize the assembly of MDA5 2CARD oligomers in the absence of polyUb. Multi‐signal sedimentation velocity analysis indicates that Ub₄ binds to RIG‐I 2CARD with a 3:4 stoichiometry and cooperatively induces formation of an RIG‐I 2CARD tetramer. In contrast, Ub₄ and Ub₇ interact with MDA5 2CARD weakly and form complexes with 1:1 and 2:1 stoichiometries but do not induce 2CARD oligomerization. In the absence of polyUb, MDA5 2CARD self‐associates to forms large oligomers in a concentration‐dependent manner. Thus, RIG‐I and MDA5 2CARD assembly processes are distinct. MDA5 2CARD concentration‐dependent self‐association, rather than polyUb binding, drives oligomerization and MDA5 2CARD forms oligomers larger than tetramer. We propose a mechanism where MDA5 2CARD oligomers, rather than a stable tetramer, function to nucleate MAVS polymerization.