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. 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

Jie Zheng  1 , Hui Yee Yong  2 , Nantika Panutdaporn  1 , Chuanfa Liu  1 , Kai Tang  1 , Dahai Luo  3
Affiliations

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

Jie Zheng et al. Nucleic Acids Res. 2015 Jan.

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.

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Figures

Figure 1.
Figure 1.
Apo states of RIG-I and MDA5. (A) Workflow of the differential HDX-MS experiments. (a) Differential HDX studies consisted of multiple experiments, e.g. apo protein, protein+RNA complex and protein+RNA&ATP. (b) Differential protein complexes were incubated with deuterated solvent at various time points under native conditions, followed by quenching (pH 2.4, 0°C) to terminate the HDX reactions. (c) Protein complexes were immediately digested by pepsin and highly reproducible peptides were separated by liquid chromatography under quenching conditions. (d) Mass spectrometry was then used to identify and characterize the deuterium-incorporated peptides. Differential HDX behavior was acquired in isotopic distribution for each peptide across various HDX time points. (e) The HDX Workbench provided the statistical analyses and graphical representations of the HDX-MS data (52). (B) Schematic representation of full length and truncated form of RIG-I and MDA5, displaying CTD (light orange), Pincer (light red), HEL1 (light green), HEL2 (light purple), HEL2i (light blue) and tandem CARDs (gray). RIG-IΔCARDs and MDA5ΔCARDs are illustrated in the dashed box. (C) The auto-inhibited conformation of apo RIG-I is maintained by the CARD2 and HEL2i domain (structure model based on duck RIG-I apo enzyme, PDB: 4a2w). Deuterium uptake plots of the CARD2 latch peptide (Y103-114) and the HEL2i gate peptides (Y566-574 and V522-539) from RIG-I and RIG-IΔCARDs are shown. The data are plotted as percent deuterium uptake versus time on a logarithmic scale. Red, blue plots represent the RIG-I (RIG-IΔCARDs) apoenzyme and the 3p10L bound state, respectively. (D) The CARD2-HEL2i interface is absent in the MDA5 apoenzyme (structure model based on the duck RIG-I CARDs, PDB: 4a2w and human MDA5ΔCARDs, PDB: 4gl2). Deuterium uptake plots of the CARD2 peptide (T100-114) and the HEL2i peptide (F630-642) from MDA5 and MDA5ΔCARDs are shown. The data are plotted as percent deuterium uptake versus time on a logarithmic scale. Red and purple plots represent the MDA5 (MDA5ΔCARDs) apoenzyme and + polyIC state, respectively. (E) The RIG-I CARD2 peptides Y103-114 and L160-175 follow EX1 kinetics (in red) with two distinct mass isotopic distributions. In contrast, the RIG-I CARD2 peptide Y108-128 and the MDA5 CARD2 peptides T100-115 and D183-193 follow EX2 kinetics (in black) in which only one mass isotopic distribution is present.
Figure 2.
Figure 2.
Characteristics of RNA and ATP binding to RIG-I. (A) Characteristic HDX profiles of RIG-I peptides induced by different RNA ligands. Deuterium uptake for peptides (Y103-114, Y566-574, F293-310 and F882-891) after a 1-h exposure to deuterated solvent. Asterisks indicate significant differences based on one-way ANOVA/Tukey between apo and +RNA states (* = P < 0.04; ** = P < 0.005). Isotopic distributions of selected peptides are shown for indicated states. (B) Deuterium uptake plots of RIG-I CTD capping loop. Deuterium uptake plots of peptide (V843-856) in RIG-I and RIG-IΔCARDs are shown. The data are plotted as percent deuterium uptake versus time on a logarithmic scale. Red, blue and purple plots represent RIG-I apo enzyme, +3p10L and +polyIC state, respectively. The right panel illustrates how the CTD binds 3p10L (end capping) and polyIC (end capping and internal binding) differently. (C) Characterization of ATP analog effects on RIG-I domain peptides. Deuterium uptake for peptides (Y103-114, Y566-574, F293-310, F633-645, Y715-724 and F882-891) in different ATP analog binding states after a 5-min exposure to deuterated solvent. Asterisks indicate significant differences based on one-way ANOVA/Tukey (* = P < 0.05; ** = P < 0.005). Isotopic distributions of selected peptides are shown for indicated states.
Figure 3.
Figure 3.
Structural basis of cooperative MDA5 binding to long duplex RNA. (A) The front view: deuterium uptake plots of MDA5 peptides involved in inter-molecular interactions (F374-391, K398-409, F630-642, A745-762, V763-785 and I940-959). The data are plotted as percentage deuterium uptake versus time on the logarithmic scale. Red plots indicate MDA5 apo enzyme and purple plots represent +polyIC state, showing that these surface peptides are more protected against HDX in the presence of polyIC. These peptides are colored in blue and mapped to MDA5 front surface. The HEL2i surface loop N643-674 (in gray), which is truncated in the reported crystal structure, is also modeled according to its HDX profile. (B) The back view: deuterium uptake plots of the peptides involved in inter-molecular interactions (T489-512, F630642, R850-868, Q877-902 and L913-938). (C) A model displays cooperative binding of three neighboring MDA5 monomers to the long dsRNA. This model is constructed based on our HDX data taking the previous cross-linking experiments into consideration (24,27,42). The front and back interface peptides of the central MDA5 monomer (n) are colored in blue and light blue according to (A) and (B). The interfaces of two neighboring MDA5 monomers (n−1 and n+1) are highlighted as well. See also the Supplementary Movie S1 on the cooperative binding of MDA5 to duplex RNA.
Figure 4.
Figure 4.
A model of the RIG-I and MDA5 activation pathways. (A) In the resting state, RIG-I adopts an auto-inhibited conformation via CARD2-HEL2i interaction while the HEL and CTD domains maintain a flexible and extended conformation available for sensing viral RNA. Binding of 3p10L triggers a drastic structural transformation resulted in a ring-shape architecture in which CTD captures the terminus of the duplex RNA and HEL clamps the backbone. RIG-I then forms a semi-opened conformation in which tandem CARDs are partially released from the gate motif on HEL2i. ATP binding results in complete disengagement of CARDs away from HEL2i (7,19). Upon binding to polyIC, RIG-I adopts a dynamic conformation in which both CTD and HEL bind to polyIC and CARDs dissociate from HEL2i. There is no cooperativity between individual RIG-I proteins. (B) MDA5 apoenzyme adopts an open and flexible conformation. Upon binding to polyIC, MDA5 proteins establish cooperative binding with neighboring molecules on the helical dsRNA backbone through their HEL-CTD rings. Five MDA5 molecules are estimated to form a repetitive unit (7,27,42). In this state, tandem CARDs are loosely connected to the HEL-CTD via a long linker. There is very little conformational change upon ATP binding and hydrolysis. (C) K63-poly ubiquitin chains assist RIG-I and MDA5 CARDs tetramerization on mitochondrial outer membrane and MAVS oligomerization then occurs via MAVS-CARD:RLR-CARDs interactions (6,30,33,36).
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