Structural basis for ubiquitin-mediated antiviral signal activation by RIG-I

RIG-I protein recognizes viral duplex RNA with a 5′-triphosphate group, activating innate immune responses; a crystal structure of its tetrameric CARD signalling domain reveals that non-covalently linked ubiquitin chains stabilize the tetramer in a ‘lock-washer’ structure that serves as a signalling platform for the recruitment and activation of MAVS. The protein RIG-I recognizes viral RNA sequences, activating innate immune responses. This protein is known to make both covalent and non-covalent interactions with K63-linked ubiquitin chains. It has remained unclear, however, how these ubiquitin modifications regulate the activity of 2CARD, the signalling domain of RIG-I, to allow its release in response to viral RNA and subsequent interaction with the downstream signalling molecule MAVS. Sun Hur and colleagues now present the crystal structure of 2CARD tetramer bound by three K63-ubiquitin chains. They find that the non-covalently linked ubiquitin chains bridge adjacent subunits of 2CARD and the covalently linked chains further stabilize this tetramer, which resembles a 'lock-washer' with its surface serving as a signalling platform for recruitment and activation of MAVS. Ubiquitin (Ub) has important roles in a wide range of intracellular signalling pathways. In the conventional view, ubiquitin alters the signalling activity of the target protein through covalent modification, but accumulating evidence points to the emerging role of non-covalent interaction between ubiquitin and the target. In the innate immune signalling pathway of a viral RNA sensor, RIG-I, both covalent and non-covalent interactions with K63-linked ubiquitin chains (K63-Ub n ) were shown to occur in its signalling domain, a tandem caspase activation and recruitment domain (hereafter referred to as 2CARD)1,2. Non-covalent binding of K63-Ub n to 2CARD induces its tetramer formation, a requirement for downstream signal activation3. Here we report the crystal structure of the tetramer of human RIG-I 2CARD bound by three chains of K63-Ub2. 2CARD assembles into a helical tetramer resembling a ‘lock-washer’, in which the tetrameric surface serves as a signalling platform for recruitment and activation of the downstream signalling molecule, MAVS. Ubiquitin chains are bound along the outer rim of the helical trajectory, bridging adjacent subunits of 2CARD and stabilizing the 2CARD tetramer. The combination of structural and functional analyses reveals that binding avidity dictates the K63-linkage and chain-length specificity of 2CARD, and that covalent ubiquitin conjugation of 2CARD further stabilizes the Ub–2CARD interaction and thus the 2CARD tetramer. Our work provides unique insights into the novel types of ubiquitin-mediated signal-activation mechanism, and previously unexpected synergism between the covalent and non-covalent ubiquitin interaction modes.