Immunity Zheng Jie's team reveals K63-polyUb-mediated MDA5-MAVS antiviral signaling pathway assembly program and activation mechanism

Editor | XI MDA5 is an intracellular allogeneic RNA monitoring protein and is an important member of the RIG-I-like receptor family (RLRs)
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MDA5 is mainly involved in regulating the immune response caused by RNA viruses such as coronavirus, picornavirus, flavivirus and hepatitis D virus, and is an important barrier to natural immunity
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MDA5 can recognize endogenous alu RNA and the double-stranded RNA released by mitochondria in autoimmune diseases
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There are three members in the RLRs family: RIG-I, MDA5 and LGP2.
The N-terminus of RLRs and MDA5 both possess tandem CARDs domains
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CARDs belong to the Death Domain superfamily, which can recruit MAVS through CARD-CARD homotype interaction, and ultimately promote the activation of type I interferon (IFN) pathway
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Ubiquitination is an important post-translational modification in eukaryotes
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Ubiquitination can be reversibly regulated by E1, E2 and E3 enzymes and DUBs
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Ubiquitin can be modified by homoubiquitin molecules and assembled into polyubiquitin chains, including M1, K11, K27, K48, and K63 and other differently connected polyubiquitin chains
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Among them, the K63-linked polyubiquitin chain (K63-polyUb) plays a key role in regulating RLRs signaling pathway [1]
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Previous studies have found that short-chain K63-polyUb can effectively promote the oligomerization of RIG-ICARDs through covalent anchoring and non-covalent anchoring [2,3]
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The formed heterotetrameric complexes (K63-polyUb-RIG-ICARDs) can activate MAVSCARD oligomerization and form the core of MAVS fibers [2,3]
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However, how K63-polyUb regulates the assembly of MDA5 CARDs and the molecular mechanism of recruitment and activation of MAVS CARD is an unresolved scientific problem
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Although K63-polyUb is required for MDA5-mediated activation of MAVS [4], no MDA5 CARDs covalently modified by K63-polyUb have been found
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This implies that the non-anchored K63-polyUb plays an important role in MDA5-MAVS signal transmission
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On October 12, 2021, Zheng Jie’s team from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences published the Ordered assembly of the cytosolic RNA-sensing MDA5-MAVS signaling complex via binding to unanchored K63-linked poly-ubiquitin online in Immunity as a Research Article chains, revealing the molecular mechanism by which long chains rather than anchoring K63-polyUb promote MDA5-MAVS assembly procedures and signal transmission
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First, the researchers established a K63-, K48-linked biochemical synthesis platform for ubiquitin chains, and prepared K63-polyUbn (2≤n≤14) of different lengths (Figure 1)
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Through Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS), the researchers found that the protection degree of hydrogen deuterium exchange of MDA5CARDs and RIG-ICARDs depends on K63-polyUbn of different lengths (MDA5: n≥8; RIG- I: n≥3) does not depend on K48-polyUbn (n≥10); and the protection strength increases specifically as the length of K63-polyUb increases
.

Figure 1: HDX-MS analysis of the effect of K63-polyUb (2≤n≤14) on RLR CARDs oligomerization The structure of the complex of MDA5CARDs and K63-polyUb13 with a rate of 3.
3 Å
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This is also the first cryo-EM structure of MDA5CARDs with near-atomic resolution
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Eight Ub molecules surround a left-handed spiral, cyclic CARDs tetramer, which includes five unique Ub-CARD interaction interfaces and seven CARD1-2 unique intramolecular and intermolecular interaction interfaces (Ia: Ib, IIa:IIb, and IIIa:IIIb) (Figure 2)
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So how does the MDA5CARDs-K63-polyUbn heterotetramer recruit its downstream signaling protein MAVS? The researchers further analyzed Cryo-EM to obtain a “bottom-up” left-handed spiral MDA5CARDs-MAVSCARD complex tethered by a long chain K63-polyUb11 with a resolution of 3.
2 Å.
Stacked on top of the tetramer of MDA5CARDs tethered by eight Ub molecules
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The MDA5CARD2 and MAVSCARD epitopes include three unique intermolecular isotype interfaces (Ia:Ib, IIa:IIb, and IIIa:IIIb)
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Mutations in the amino acid sites of the interaction interface can cause defects in the activation of the IFN signaling pathway in the cell
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Figure 2: Single-particle cryo-electron microscopy shows the assembly mechanism of MDA5-MAVS CARD-CARD mediated by K63-polyUbn (n≥8).
At the same time, researchers used biomacromolecule hydrogen deuterium exchange mass spectrometry technology to in-depth characterize the full-length MDA5 in solution Under the dynamic conformational changes and signal transduction mechanism of interaction with K63-polyUb10
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Hydrogen-deuterium exchange mass spectrometry proved for the first time that CARDs of human MDA5 full-length protein are in an open conformation in the initial state and can bind to the long chain K63-polyUb10
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However, in early studies, hydrogen-deuterium exchange mass spectrometry has proved that RIG-ICARDs are in a closed conformation in the initial state [5,6]
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This also directly proves the huge difference in the conformation of RIG-I and MDA5 CARDs in solution
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Second, the researchers further discovered that the stability of the K63-polyUb10 tethered MDA5CARDs complex in solution is allosterically regulated by MDA5's RNA-dependent ATPase activity: MDA5 stimulated by RNA can bind and hydrolyze ATP, and the energy produced by its binding It can remotely stabilize the assembly of K63-polyUb10-MDA5CARDs to continuously transmit downstream activation signals; on the contrary, although MDA5 not stimulated by RNA can also bind to the long chain K63-polyUb10, ATP can depolymerize it and prevent unnecessary MDA5.
Activate (Figure 3)
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These evidences indicate that the effective recognition of RNA by MDA5 near the mitochondrial membrane containing high concentrations of ATP is important for remote regulation of the stability of the K63-polyUb10-MDA5CARDs complex and the activation of MAVS.

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Figure 3: HDX-MS analysis of the dynamic conformational changes and signal transduction mechanism of full-length MDA5 under the action of its recognition ligand or substrate (dsRNA/ATP/K63-polyUb).
Deuterium exchange mass spectrometry and cryo-electron microscopy techniques have found that the long chain, non-anchored K63-polyUb is similar to a "molecular bridge", which promotes the assembly of MDA5CARDs tetramers and makes them form an excited conformation to recruit further oligomerization and further oligomerization of downstream MAVSCARDs.
Activate (Figure 3)
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In the activated state, MDA5 can bind and hydrolyze ATP, and remotely promote the stability of CARDs-K63-polyUb10 to continuously activate MAVS
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This research fills up the gaps in the study of MDA5 pathway activation and signal transduction, and further reveals the immunological function of long-chain, non-anchored K63-polyUb as an endogenous agonist in the cell, in order to understand the molecular diversity of ubiquitin in anti-RNA The role of viral innate immune signal transduction and regulation provides new clues
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Song Bin, a postdoctoral fellow at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and Chen Yun, an American NIH Research Associate, are the co-first authors of the paper
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Researcher Zheng Jie from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences is the corresponding author of the paper .

This work was strongly supported by Professor Luo Dahai and Professor Wu Bin from Nanyang Technological University in Singapore, Professor Patrick Griffin from the American Scripps Research Institute, and researcher Luo Cheng and researcher Zhang Naixia from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences
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Postdoctoral Recruitment The Zheng Jie Laboratory of Shanghai Institute of Materia Medica, Chinese Academy of Sciences has long-term combined with biological macromolecule hydrogen deuterium exchange mass spectrometry technology to cross-solve the mechanism of major diseases caused by abnormal changes in protein (enzyme) dynamics, focusing on RNA natural immune pattern recognition.
The body’s endogenous and exogenous ligand recognition and signal transduction mechanisms, as well as the mechanism of autoimmune diseases
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The laboratory is now recruiting 2 postdoctoral fellows in biochemistry, molecular immunology or biological mass spectrometry.

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For more information, please refer to the website: http:// Original link: https://doi.
org/10.
1016/j.
immuni.
2021.
09.
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