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

• Main Authors: Liu, Chuan-Fa, Zheng, Jie, Yong, Hui Yee, Panutdaporn, Nantika, Tang, Kai, Luo, Dahai
• Other Authors: Lee Kong Chian School of Medicine (LKCMedicine)
• Format: Article
• Language: English
• Published: 2015
• Subjects: DRNTU::Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/103974; http://hdl.handle.net/10220/24589
• Institution: Nanyang Technological University

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.