A loosened gating mechanism of RIG-I leads to autoimmune disorders

Abstract

DDX58 encodes RIG-I, a cytosolic RNA sensor that ensures immune surveillance of nonself RNAs. Individuals with RIG-IE510V and RIG-IQ517H mutations have increased susceptibility to Singleton-Merten syndrome (SMS) defects, resulting in tissue-specific (mild) and classic (severe) phenotypes. The coupling between RNA recognition and conformational changes is central to RIG-I RNA proofreading, but the molecular determinants leading to dissociated disease phenotypes remain unknown. Herein, we employed hydrogen/deuterium exchange mass spectrometry (HDX-MS) and single molecule magnetic tweezers (MT) to precisely examine how subtle conformational changes in the helicase insertion domain (HEL2i) promote impaired ATPase and erroneous RNA proofreading activities. We showed that the mutations cause a loosened latch-gate engagement in apo RIG-I, which in turn gradually dampens its self RNA (Cap2 moiety:m7G cap and N1-2-2'-O-methylation RNA) proofreading ability, leading to increased immunopathy. These results reveal HEL2i as a unique checkpoint directing two specialized functions, i.e. stabilizing the CARD2-HEL2i interface and gating the helicase from incoming self RNAs; thus, these findings add new insights into the role of HEL2i in the control of antiviral innate immunity and autoimmunity diseases.

Figure 1.

RIG-I HEL2i mutations destabilize the CARD2-HEL2i engagement and lead to increased solvent exposure. (A) Examination of IRF3 phosphorylation and IFN-β reporter assays of SMS RIG-I variants. The indicated SMS RIG-I constructs were expressed in the presence and absence of RNA treatment to examine their contributions to the induction of IFN-β responses. Data are presented as the mean values ± SEM; n = 3 independent experiments; statistics indicate the significance of differences between WT and the respective mutant determined by Student's t test: *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. N.S. denotes nonsignificant. (B) ATP hydrolysis assay of SMS RIG-I variants bound with 3p10l RNA. (Data are presented as the mean values ± SEM, n = 3 independent experiments; statistics indicate significance of differences between compared sample states determined by Student's t test: *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. N.S. denotes nonsignificant.). (C) Single amide consolidated HDX-MS profiling of apo RIG-I, RIG-I_E510V and RIG-I_Q517H. The y- and x-axes illustrate the D% and domain arrangement of RIG-I, respectively. In the lower panel, a positive value on the y-axis (ΔHDX %) represents deprotection (solvent exposure) against deuterium exchange in the corresponding region depicted along the x-axis of apo RIG-I_E510V and apo RIG-I_Q517H, as compared with apo RIG-I. (D) An apo RIG-I model is shown on the left panel, and the CARD2-HEL2i interface is highlighted, wherein the CARD2 latch peptide (103–114) is colored red. The analysis of the EX1 kinetics is shown on the right panel. It displays the MS spectra of CARD2 latch peptide (103–114) derived from the indicated complexes at the indicated on-exchange time points. The abundance of each mass population (high and low) is determined by fitting a bimodal Gauss-function to the isotopic envelope by HX express2 software. (E) CARDs partial opening rate calculated from EX1 analysis. Half-life (t_1/2) of respective partial unfolding event is determined by fitting an exponential 3P with the prediction model: a + b ×exp(c.time(min)), where a is the asymptote, b is the scale and c is the growth rate, is used to fit a curve to %D (response) and time (regressor).

Figure 2.

Profiling the conformations of RIG-I and RIG-I_Q517H by single-molecule magnetic tweezers (MT). (A) Schematic of RIG-I domains, tag engineering, and conformational profiling in MT. (B, C) Representative conformational curves of RIG-I (B) and RIG-I_Q517H (C). Three main conformational changes are marked with arrows, wherein the first is magnified in a dashed box labeled with step size. (D) Force histogram for conformational changes of RIG-I (red) and RIG-I_Q517H (blue). The three peaks obtained by Gaussian fitting are responses to the three main conformational changes in (B) and (C). (E) Schematic of the RIG-I helicase, tag engineering, and conformational profiling in MT. (F, G) Representative conformational curves of RIG-I ΔCARDs (F) and RIG-I_Q517H ΔCARDs (G). Conformational changes are marked with arrows, wherein the 0–5 pN area is magnified in a dashed box. (H) Force histogram for conformational changes of RIG-I ΔCARDs (red) and RIG-I_Q517H ΔCARDs (blue). The force peak obtained by Gaussian fitting is a response to the conformational change in (F) and (G). (I) Schematic of the RIG-I CARDs domain, tag engineering, and conformational profiling in MT. (J) Representative conformational curves of CARDs; conformational changes are marked with arrows. (K) Force histogram for conformational changes of CARDs. The force peak obtained by Gaussian fitting is a response to the conformational change in (J). (L) The size of CARD2-HEL2i dissociation of RIG-I and RIG-I_Q517H. Data were derived from <3.75 pN, wherein the first conformational change occurred. (M) The dissociation probability of CARD2-HEL2i of RIG-I and RIG-I_Q517H; results are represented from a single tether that was repeatedly stretched for at least 6 cycles. (N) Conformational changing profiles of RIG-I and RIG-I_Q517H, wherein CARD2-HEL2i dissociation, helicase unfolding and CARDs unfolding occurred sequentially upon force. Data are presented as the mean ± SD in (D), (H) and (K) and as the mean ± SEM in (L) and (M); statistics indicate the significance of differences between compared sample states determined by Student's t test: ****P ≤ 0.0001; NS denotes nonsignificant in (L) and (M).

Figure 3.

Impaired RNA proofreading capabilities associated with RIG-I_Q517H and RIG-I_E510V. (A) Examination of IRF3 phosphorylation and IFN-β reporter assays of RIG-I_E510V and RIG-I_Q517H upon respective RNA treatment. Data are presented as the mean values ± SEM; n = 3 independent experiments; statistics indicate significance of differences between WT and the respective mutant determined by Student's t test: *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001. N.S. denotes nonsignificant. (B) RNA-dependent ATPase activity profiles of the indicated protein complexes. (C) Single amide consolidated HDX-MS profiling of RIG-I, RIG-I_E510V and RIG-I_Q517H upon RNA discrimination between 3p10l and Cap2-10l. In the uppor panel, the y- and x-axes illustrate the HDX perturbations (ΔHDX%) of respective RNA binding event and domain arrangement of RIG-I or its HEL2i variants, respectively. A positive value or a negative value in the y axis (ΔHDX%) represents protection or deprotection against deuterium exchange in the corresponding region of the receptor depicted in the x axis when a RNA binding event (induced by 3p10l or Cap2-10l) takes place. In the lower panel, it displays the comparative HDX analysis (ΔΔHDX%) between 3p10l- and Cap2-10l-mediated HDX perturbations on RIG-I, RIG-I_E510V and RIG-I_Q517H. ΔΔHDX% values (ΔΔHDX = ΔHDX_{RIG-I(WT/mut)} ± _3p10l - ΔHDX_{RIG-I(WT/mut)} ± _Cap2-10l) could indicate the conformational perturbations of RIG-I or its variants upon RNA discrimination. (D) Differential single amino acid consolidation HDX data are mapped onto the full-length RNA-bound RIG-I structure model in the ribbon, as shown by the representation of ΔΔHDX profiles of RIG-I, RIG-I_E510V and RIG-I_Q517H (Supplementary Figure S5, columns (xiii, xiv and xv), and Figure S8). Percentages of deuterium differences are color-coded according to the HDX key (below). Black, regions that have no sequence coverage and include proline residues that have no amide hydrogen exchange activity; gray, no statistically significant changes between compared states; purple, duplex RNA ligand.

Figure 4.

RIG-I_Q517H extends its conformation unbiasedly upon RNA proofreading. (A) Schematic revealing the RIG-I and RIG-I_Q517H discrimination of viral and self RNAs in MT. When RIG-I and RIG-I_Q517H were captured with a low force (∼1.3 pN) in MT, HEPES buffer flowed in as the control, and then the length increase of RIG-I or RIG-I_Q517H driven by RNAs was measured. (B, C) Representative data of the length increase of RIG-I driven by 3p10l RNA (B) and RIG-I driven by Cap2-10l RNA (C), wherein the relative RIG-I length under different conditions was quantified by Gaussian fitting. (D) Schematic of the length increase of RIG-I driven by 3p10l RNA or Cap2-10l RNA. (E, F) Representative data of the length increase of RIG-I_Q517H driven by 3p10l RNA (E) and RIG-I_Q517H driven by Cap2-10l RNA (F), wherein the relative RIG-I_Q517H length under different conditions was quantified by Gaussian fitting. (G) Schematic of the length increase of RIG-I_Q517H driven by 3p10l RNA or Cap2-10l RNA. (H) Length increases in RIG-I and RIG-I_Q517H driven by 3p10l RNA or Cap2-10l RNA. Each dot is derived from repeated experiments. (I) Length increases driven by 3p10l RNA and Cap2-10l RNA. Data are presented as the mean ± SD in (B), (C), (E) and (F) and as the mean ± SEM in (H); statistics indicate significance of differences between compared sample states determined by Student's t test: **P ≤ 0.01; NS represents nonsignificant in (H).

Figure 5.

Cartoon representations of RIG-I autoimmune signaling events. In the resting state, RIG-I adopts an autoinhibited conformation via CARD2-HEL2i interaction. RIG-I can recognize nonself RNA and discriminate against self RNA, resulting in distinguished CARDs opening events. Unlike WT RIG-I, apo RIG-I_E510V and apo RIG-I_Q517H are coupled with a reduced gradient of CARD2-HEL2i engagement. RIG-I_E510V can distinguish self from nonself RNA via its HEL-CTD module and activate CARDs differently but to lesser extents than RIG-I. RIG-I_Q517H, however, fails to distinguish self from nonself RNA and results in fast CARDs unfolding events at similar unfolding rates. The subsequent K63-polyUb₉ can bind to activated CARDs and trigger CARDs oligomerizations prior to MAVS activation.