MDA5 is an allogeneic RNA monitoring protein in cells and an important member of RIG-I-like receptor family (RLRs). As an important barrier of natural immunity, it is mainly involved in regulating the immune response caused by RNA viruses. The RLRs family has three members, i.e., RIG-I, MDA5 and LGP2.

Ubiquitination is an important post-translational modification in eukaryotes. Ubiquitin can be modified by homo ubiquitin molecules and assembled into polyubiquitin chains, including M1, K11, K27, K48 and K63. Among them, K63-linked polyubiquitin chain (K63-polyUb) plays a key role in regulating RLRs signal pathway. However, how K63-polyUb regulates the assembly of ^MDA5CARDs and the molecular mechanism of recruiting and activating ^MAVSCARD remains a problem to be solved.

In a study published in Immunity, ZHENG Jie's group from Shanghai Institute of Materia Medica of the Chinese Academy of Sciences revealed the molecular mechanism that long chain, unanchored K63-polyUb promotes MDA5-MAVS assembly program and signal transduction.

Through hydrogen deuterium exchange mass spectrometry (HDX-MS), the researchers found that ^MDA5CARDs prefer to combine longer K63-polyUbn than ^RIG-ICARDs (MDA5: n ≥ 8; RIG-I: n ≥ 3), but not K48-polyUbn (n ≥ 10).

To study the assembly mechanism of oligomers of ^MDA5CARDs mediated by K63-polyUbn, they analyzed the structure of the complex of ^MDA5CARDs-K63 polyUb₁₃ with a resolution of 3.3 Angstrom by Cryo-Electron Microscope (Cryo-EM) for the first time. The structure showed eight UB molecules surround a left-handed helical, cyclic CARDs tetramer. This is also the first Cryo-EM structure of ^MDA5CARDs with near atomic resolution.

But how does the heterotetramer of ^MDA5CARDs-K63 polyUbn recruit its downstream signal protein MAVS? The researchers obtained the "bottom-up" left-handed helical ^MDA5CARDs-^MAVSCARD complex tethered by long chain K63-polyUb₁₁ with a resolution of 3.2 Angstrom by Cryo-EM analysis.

Moreover, the researchers proved for the first time that the 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 through HDX-MS. However, in early studies, it was proved by HDX-MS that ^RIG-ICARDs has a closed conformation in the initial state. This also directly proved the great difference in the conformation of cards of ^RIG-I and MDA5 in solution.

In addition, they found that the stability of K63-polyUb₁₀ tethered ^MDA5CARDs complex in solution is regulated by the RNA dependent ATPase activity of MDA5, and the effective recognition of RNA by MDA5 near mitochondrial membrane containing high concentration of ATP helps to remotely regulate the stability of K63 polyUb₁₀-^MDA5CARDs complex and helps activate MAVS.

This study provides a new clue for understanding the role of ubiquitin molecular diversity in anti-RNA virus natural immune signal transduction.