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

High-resolution HDX-MS reveals distinct mechanisms of RNA recognition and activation by RIG-I and MDA5.

Author information

1
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
2
Lee Kong Chian School of Medicine, Nanyang Technological University, 61 Biopolis Drive, Proteos Building, #07-03, 138673, Singapore.
3
Lee Kong Chian School of Medicine, Nanyang Technological University, 61 Biopolis Drive, Proteos Building, #07-03, 138673, Singapore luodahai@ntu.edu.sg.

Abstract

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.

PMID:
25539915
PMCID:
PMC4333383
DOI:
10.1093/nar/gku1329
[Indexed for MEDLINE]
Free PMC Article

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