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Assembly of Signaling Complexes01:30

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Updated: Jan 4, 2026

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study
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Oligomerization of RIG-I and MDA5 2CARD domains.

Cassie M Zerbe1, David J Mouser1, James L Cole1,2

  • 1Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut.

Protein Science : a Publication of the Protein Society
|November 8, 2019
PubMed
Summary

The retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA5) have distinct signaling complex assembly mechanisms. MDA5 CARDs self-associate concentration-dependently, unlike RIG-I CARDs which form tetramers upon polyubiquitin binding.

Keywords:
K63-linked polyubiquitinRIG-I-like receptorsanalytical ultracentrifugationinnate immunitymulti-signal sedimentation velocity

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Area of Science:

  • Immunology
  • Molecular Biology
  • Biochemistry

Background:

  • The innate immune system relies on retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) for viral RNA recognition.
  • RIG-I and melanoma differentiation-associated protein 5 (MDA5) are key RLRs, each possessing N-terminal caspase activation and recruitment domains (2CARD).
  • RLR activation leads to MAVS oligomerization, downstream signaling, and interferon induction, but the MDA5 2CARD complex assembly remains unclear.

Purpose of the Study:

  • To investigate and compare the polyubiquitin (polyUb) binding and oligomerization properties of MDA5 2CARD and RIG-I 2CARD.
  • To elucidate the distinct mechanisms governing MDA5 2CARD and RIG-I 2CARD signaling complex formation.

Main Methods:

  • Sedimentation velocity analytical ultracentrifugation was employed to analyze protein-ligand interactions.
  • Comparative analysis of MDA5 2CARD and RIG-I 2CARD binding to polyubiquitin chains (Ub4, Ub7).
  • Characterization of MDA5 2CARD oligomerization in the absence of polyubiquitin.

Main Results:

  • RIG-I 2CARD binds Ub4 with 3:4 stoichiometry, cooperatively forming a tetramer.
  • MDA5 2CARD interacts weakly with Ub4 and Ub7 (1:1 and 2:1 stoichiometries) without inducing oligomerization.
  • MDA5 2CARD undergoes concentration-dependent self-association, forming oligomers larger than tetramers in the absence of polyubiquitin.

Conclusions:

  • RIG-I and MDA5 2CARD exhibit distinct assembly processes.
  • MDA5 2CARD oligomerization is driven by self-association, not polyubiquitin binding.
  • Large MDA5 2CARD oligomers, rather than a stable tetramer, likely nucleate MAVS polymerization.