Oligomerization of RIG-I and MDA5 2CARD domains

Abstract

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.

Keywords: K63-linked polyubiquitin; RIG-I-like receptors; analytical ultracentrifugation; innate immunity; multi-signal sedimentation velocity.

Figure 1

Domain structures of retinoic acid‐inducible gene I (RIG‐I) and melanoma differentiation‐associated protein 5 (MDA5). (a) The 2CARD domain is shown in red (CARD 1) and blue (CARD 2) and the helicase and C‐terminal domains are shown in gray. (b) Sequence alignment of RIG‐I and MDA5 2CARD showing polyUb binding sites. Residues in green on RIG‐I 2CARD interact with polyUb in the RIG‐I 2CARD crystal structure.20 For MDA5, residues aligned to polyUb‐binding residues of RIG‐I are shown in green and conserved amino acids are indicated by asterisks

Figure 2

Multi‐signal sedimentation velocity analysis of retinoic acid‐inducible gene I (RIG‐I) like receptor (RLR) 2CARD binding to K63‐linked Ub₄. c(s) Distributions of: (a) 5 μM retinoic acid‐inducible gene I (RIG)‐I 2CARD and 5 μM Ub₄ alone, (b) mixture of 5 μM RIG‐I 2CARD and 5 μM Ub₄, (c) mixture of 5 μM RIG‐I and 20 μM Ub₄, (d–f) same as a‐c, but samples contain melanoma differentiation‐associated protein 5 (MDA5) 2CARD and Ub₄. Peaks are labeled with the experimental molecular weights and the 2CARD: Ub₄ stoichiometries. Data were collected at 20°C and 45,000 RPM using absorbance at 280 nm and Rayleigh interferometry. Note that the peak for free MDA5 2CARD shifts towards the Ub₄ peak in panels (e) and (f). This likely represents an artifact arising from the c(s) deconvolution

Figure 3

Multi‐signal sedimentation velocity analysis of melanoma differentiation‐associated protein 5 (MDA5) 2CARD binding to K63‐linked Ub₇. C(s) distributions of (a) of 5 μM MDA5 2CARD and Ub₇ alone. (b) Mixture of 5 μM MDA5 2CARD and 10 μM Ub₇. Peaks are labeled with the experimental molecular weights and the 2CARD: Ub₇ stoichiometries. Data were collected at 20°C and 45,000 RPM using absorbance at 280 nm and Rayleigh interferometry. Note that the peak for free MDA5 2CARD shifts towards the Ub₇ peak in panels E and F. This likely represents an artifact arising from the c(s) deconvolution

Figure 4

Concentration‐dependent self‐association of melanoma differentiation‐associated protein 5 (MDA5) 2CARD. C(s) distributions of samples are presented for samples at loading concentrations of 0.1, 0.25, 1.0 and 2.5 mg/ml MDA5 2CARD. Data are normalized by area. Monomeric MDA5 2CARD was concentrated to the indicated concentration in AU20 buffer. Data were collected at 20°C and 50,000 RPM using Rayleigh interferometry. The aggregate populations are: 3.5, 17, 25 and 64% for the 0.1, 0.25, 1, and 2.5 mg/ml samples, respectively