title: High-resolution HDX-MS reveals distinct mechanisms of RNA recognition and activation by RIG-I and MDA5.
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.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
authors.forename: Jie
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Lee Kong Chian School of Medicine, Nanyang Technological University, 61 Biopolis Drive, Proteos Building, #07-03, 138673, Singapore.
authors.forename: Hui Yee
authors.initials: HY
authors.lastname: Yong
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
authors.forename: Nantika
authors.initials: N
authors.lastname: Panutdaporn
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
authors.forename: Chuanfa
authors.initials: C
authors.lastname: Liu
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
authors.forename: Kai
authors.initials: K
authors.lastname: Tang
authors.affiliation: Lee Kong Chian School of Medicine, Nanyang Technological University, 61 Biopolis Drive, Proteos Building, #07-03, 138673, Singapore luodahai@ntu.edu.sg.
authors.forename: Dahai
authors.initials: D
authors.lastname: Luo
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000818:Animals; D001051:Apoenzymes / Q000737:chemistry; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000737:chemistry* / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D006801:Humans; D000072640:Interferon-Induced Helicase, IFIH1; D013058:Mass Spectrometry; D051379:Mice; D008958:Models, Molecular; D011070:Poly I-C / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D011971:Receptors, Immunologic
chemical_list: D001051:Apoenzymes; D011971:Receptors, Immunologic; D012313:RNA; D000255:Adenosine Triphosphate; C452820:RIGI protein, human; C510592:Ifih1 protein, mouse; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; D000072640:Interferon-Induced Helicase, IFIH1; D011070:Poly I-C