High-resolution HDX-MS Reveals Distinct Mechanisms of RNA Recognition and Activation by RIG-I and MDA5

Nucleic Acids Res. 2015 Jan;43(2):1216-30. doi: 10.1093/nar/gku1329. Epub 2014 Dec 24.


RIG-I and MDA5 are the major intracellular immune receptors that recognize viral RNA species and undergo a series of conformational transitions leading to the activation of the interferon-mediated antiviral response. However, to date, full-length RLRs have resisted crystallographic efforts and a molecular description of their activation pathways remains hypothetical. Here we employ hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) to probe the apo states of RIG-I and MDA5 and to dissect the molecular details with respect to distinct RNA species recognition, ATP binding and hydrolysis and CARDs activation. We show that human RIG-I maintains an auto-inhibited resting state owing to the intra-molecular HEL2i-CARD2 interactions while apo MDA5 lacks the analogous intra-molecular interactions and therefore adopts an extended conformation. Our work demonstrates that RIG-I binds and responds differently to short triphosphorylated RNA and long duplex RNA and that sequential addition of RNA and ATP triggers specific allosteric effects leading to RIG-I CARDs activation. We also present a high-resolution protein surface mapping technique that refines the cooperative oligomerization model of neighboring MDA5 molecules on long duplex RNA. Taken together, our data provide a high-resolution view of RLR activation in solution and offer new evidence for the molecular mechanism of RLR activation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Allosteric Regulation
  • Animals
  • Apoenzymes / chemistry
  • DEAD Box Protein 58
  • DEAD-box RNA Helicases / chemistry*
  • DEAD-box RNA Helicases / metabolism
  • Deuterium Exchange Measurement
  • Humans
  • Interferon-Induced Helicase, IFIH1
  • Mass Spectrometry
  • Mice
  • Models, Molecular
  • Poly I-C / metabolism
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Tertiary
  • RNA / chemistry
  • RNA / metabolism*


  • Apoenzymes
  • RNA
  • Adenosine Triphosphate
  • DDX58 protein, human
  • Ifih1 protein, mouse
  • DEAD Box Protein 58
  • DEAD-box RNA Helicases
  • Interferon-Induced Helicase, IFIH1
  • Poly I-C