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

Abstract

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-Ubn) 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-Ubn 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.

Structure determination of helical filaments by solid-state NMR spectroscopy

“…Each protomer is in contact with six other protomers via three interfaces denoted Ia/Ib, IIa/ IIb, and IIIa,IIIb (Fig. 6 A-D), which are preserved in death domain complexes determined to date (2,21,36). The IIIa/IIIb interface mediates the intrastrand contacts that form the lefthanded 1-start helix.…”

The lock-washer: a reconciliation of the RIG-I activation models

“…Although such conflicting reports seem to propose vastly different models of RIG-I activation, an elegent study published in Nature by Peisley et al [9] uses biochemical and structural studies to reconcile the different models and they finally offer a unified understanding of RIG-I receptor activation. They resolved the crystal structure of human RIG-I 2-CARD in complex with K63-ubiquitin at 3.7 Å.…”

“…In the current study, the authors show that the assembly and stability of the tetramer and its IFN-β signaling potential are dependent on several intermolecular and intramolecular CARD interactions by generating mutants on different interaction surfaces and analyzing their tetramer formation and IFN-β induction abilities. MAVS filament formation assays indicate that the helical tetrameric structure of RIG-I 2-CARD serves as the platform for MAVS-CARD filament assembly, with the top surface of the second CARD as the primary site for MAVS recruitment [9]. The second pertinent question addressed is how the interaction between ubiquitin and 2-CARD contributes to downstream signaling?…”

RNF122 suppresses antiviral type I interferon production by targeting RIG-I CARDs to mediate RIG-I degradation

“…The RIG-I-like receptor RIG-I is a cytoplasmic viral RNA sensor that triggers the signal to induce type I IFNs and proinflammatory cytokine production in response to viral infection. RIG-I protein contains tandem caspase-recruitment domains (CARDs) at N termini, which mediate downstream signaling; a central DExD/H helicase domain with an ATP-binding motif; and a C-terminal region known as the RNA-binding domain (3)(4)(5)(6)(7). Upon binding of pathogenic RNA to the helicase domain, RIG-I undergoes a conformational change and is recruited to the mitochondrial antiviral signaling (MAVS) adaptor (8).…”