The intrinsically disordered CARDs‐Helicase linker in RIG‐I is a molecular gate for RNA proofreading
2022; Springer Nature; Volume: 41; Issue: 10 Linguagem: Inglês
10.15252/embj.2021109782
ISSN1460-2075
AutoresBrandon Schweibenz, Swapnil C. Devarkar, Mihai Solotchi, Candice Craig, Jie Zheng, Bruce D. Pascal, Samantha Gokhale, Ping Xie, Patrick R. Griffin, Smita S. Patel,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoArticle19 April 2022Open Access Transparent process The intrinsically disordered CARDs-Helicase linker in RIG-I is a molecular gate for RNA proofreading Brandon D Schweibenz Brandon D Schweibenz orcid.org/0000-0003-1532-4502 Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, USA Graduate Program in Biochemistry, Rutgers University, Piscataway, NJ, USA Contribution: Conceptualization, Data curation, Formal analysis, Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Swapnil C Devarkar Swapnil C Devarkar orcid.org/0000-0002-9271-243X Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, USA Graduate Program in Biochemistry, Rutgers University, Piscataway, NJ, USA Contribution: Conceptualization, Investigation, Writing - review & editing Search for more papers by this author Mihai Solotchi Mihai Solotchi orcid.org/0000-0003-3924-1154 Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, USA Cell and Development Biology, Rutgers University, Piscataway, NJ, USA Contribution: Investigation Search for more papers by this author Candice Craig Candice Craig orcid.org/0000-0003-0248-2149 Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, USA Graduate Program in Biochemistry, Rutgers University, Piscataway, NJ, USA Contribution: Investigation Search for more papers by this author Jie Zheng Jie Zheng Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, USA Search for more papers by this author Bruce D Pascal Bruce D Pascal Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, USA Contribution: Investigation Search for more papers by this author Samantha Gokhale Samantha Gokhale orcid.org/0000-0003-0554-099X Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ, USA Cellular and Molecular Pharmacology, Rutgers University, Piscataway, NJ, USA Contribution: Investigation Search for more papers by this author Ping Xie Ping Xie orcid.org/0000-0002-7338-3730 Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ, USA Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA Contribution: Supervision, Investigation, Writing - review & editing Search for more papers by this author Patrick R Griffin Patrick R Griffin Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, USA Department of Integrative Structural and Computational Biology, Jupiter, FL, USA Contribution: Supervision, Writing - review & editing Search for more papers by this author Smita S Patel Corresponding Author Smita S Patel [email protected] orcid.org/0000-0002-2523-4933 Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, USA Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Brandon D Schweibenz Brandon D Schweibenz orcid.org/0000-0003-1532-4502 Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, USA Graduate Program in Biochemistry, Rutgers University, Piscataway, NJ, USA Contribution: Conceptualization, Data curation, Formal analysis, Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Swapnil C Devarkar Swapnil C Devarkar orcid.org/0000-0002-9271-243X Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, USA Graduate Program in Biochemistry, Rutgers University, Piscataway, NJ, USA Contribution: Conceptualization, Investigation, Writing - review & editing Search for more papers by this author Mihai Solotchi Mihai Solotchi orcid.org/0000-0003-3924-1154 Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, USA Cell and Development Biology, Rutgers University, Piscataway, NJ, USA Contribution: Investigation Search for more papers by this author Candice Craig Candice Craig orcid.org/0000-0003-0248-2149 Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, USA Graduate Program in Biochemistry, Rutgers University, Piscataway, NJ, USA Contribution: Investigation Search for more papers by this author Jie Zheng Jie Zheng Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, USA Search for more papers by this author Bruce D Pascal Bruce D Pascal Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, USA Contribution: Investigation Search for more papers by this author Samantha Gokhale Samantha Gokhale orcid.org/0000-0003-0554-099X Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ, USA Cellular and Molecular Pharmacology, Rutgers University, Piscataway, NJ, USA Contribution: Investigation Search for more papers by this author Ping Xie Ping Xie orcid.org/0000-0002-7338-3730 Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ, USA Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA Contribution: Supervision, Investigation, Writing - review & editing Search for more papers by this author Patrick R Griffin Patrick R Griffin Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, USA Department of Integrative Structural and Computational Biology, Jupiter, FL, USA Contribution: Supervision, Writing - review & editing Search for more papers by this author Smita S Patel Corresponding Author Smita S Patel [email protected] orcid.org/0000-0002-2523-4933 Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, USA Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Brandon D Schweibenz1,2, Swapnil C Devarkar1,2, Mihai Solotchi1,3, Candice Craig1,2, Jie Zheng4, Bruce D Pascal4, Samantha Gokhale5,6, Ping Xie5,7, Patrick R Griffin4,8 and Smita S Patel *,1,7 1Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ, USA 2Graduate Program in Biochemistry, Rutgers University, Piscataway, NJ, USA 3Cell and Development Biology, Rutgers University, Piscataway, NJ, USA 4Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, USA 5Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ, USA 6Cellular and Molecular Pharmacology, Rutgers University, Piscataway, NJ, USA 7Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA 8Department of Integrative Structural and Computational Biology, Jupiter, FL, USA *Corresponding author. Tel: +1 732-235-3372; E-mail: [email protected] The EMBO Journal (2022)41:e109782https://doi.org/10.15252/embj.2021109782 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The innate immune receptor RIG-I provides a first line of defense against viral infections. Viral RNAs are recognized by RIG-I's C-terminal domain (CTD), but the RNA must engage the helicase domain to release the signaling CARD (Caspase Activation and Recruitment Domain) domains from their autoinhibitory CARD2:Hel2i interactions. Because the helicase itself lacks RNA specificity, mechanisms to proofread RNAs entering the helicase domain must exist. Although such mechanisms would be crucial in preventing aberrant immune responses by non-specific RNAs, they remain largely uncharacterized to date. This study reveals a previously unknown proofreading mechanism through which RIG-I ensures that the helicase engages RNAs explicitly recognized by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs-Helicase Linker (CHL), which connects the CARDs to the helicase subdomain Hel1. CHL uses its negatively charged regions to antagonize incoming RNAs electrostatically. In addition to this RNA gating function, CHL is essential for stabilization of the CARD2:Hel2i interface. Overall, we uncover that the CHL and CARD2:Hel2i interface work together to establish a tunable gating mechanism that allows CTD-chosen RNAs to bind the helicase domain, while at the same time blocking non-specific RNAs. These findings also indicate that CHL could represent a novel target for RIG-I-based therapeutics. Synopsis To release its autoinhibitory conformation, the innate immune receptor RIG-I helicase domain must bind RNA, but it is not fully understood how specificity for viral RNA is ensured. Here, the CARDs-Helicase linker (CHL) is identified as a regulatory element that functions together with the CARD2:Hel2i interface to establish a tunable gating mechanism for self/non-self discrimination. The ~56 aa CARDs-Helicase linker (CHL) is a regulatory unstructured region in RIG-I CHL stabilizes the autoinhibitory CARD2:Hel2i interface to keep RIG-I in a signaling-silent state CHL shields the helicase and electrostatically competes with incoming RNAs CHL and CARD2:Hel2i interface synergistically block self RNAs from activating RIG-I Introduction RIG-I (Retinoic Acid Inducible Gene-I) is an innate immune receptor responsible for surveilling the cytoplasm for viral RNAs (Yoneyama et al, 2005). RIG-I recognizes short blunt-ended double-stranded (ds) RNAs with 5'-triphosphate (5'ppp), 5'-diphosphate (5'pp), and 5'-m7G cap as PAMPs (pathogen-associated molecular pattern) (Jiang et al, 2011; Goubau et al, 2014; Schuberth-Wagner et al, 2015; Devarkar et al, 2016). Such PAMP features are not present in endogenous RNAs but found in many viral RNA genomes and most replication intermediates of negative-strand and positive-strand RNA viruses (Stumper et al, 2005; Kato et al, 2006; Rehwinkel et al, 2010; Schuberth-Wagner et al, 2015; Devarkar et al, 2016; Hu et al, 2017). Upon recognizing viral RNA PAMPs, RIG-I initiates a signaling cascade that culminates in type I interferon response, efficiently controlling viral infections (Yoneyama et al, 2004; Stumper et al, 2005; Poeck et al, 2010). Structural studies show that RIG-I has flexibly linked domains that can switch between active and inactive states to respond appropriately to viral RNAs (Fig 1A) (Civril et al, 2011; Jiang et al, 2011; Kowalinski et al, 2011; Luo et al, 2011, 2012; Devarkar et al, 2016). The core helicase domain contains two helicase subdomains, Hel1 and Hel2, and a nested Hel2i, flanked by a tandem array of signaling domain CARDs (Caspase Activation and Recruitment Domains) and a PAMP recognition C-terminal domain (CTD). When RIG-I is not bound to RNA, the helicase domain is in an open, ATPase-inactive conformation. In this conformation, the CARDs are sequestered by Hel2i through CARD2:Hel2i interactions and inactive in signaling (Fig 1A). When RIG-I fully engages the RNA in a closed helicase conformation, the induced conformational changes activate the ATPase and disrupt the CARD2:Hel2i interface to release the CARDs from their autoinhibitory interactions (Zheng et al, 2015, 2018; Dickey et al, 2019). The exposed CARDs can initiate an immune response by interacting with downstream adapter proteins (Peisley et al, 2014; Wu et al, 2014). Figure 1. The CARDs-Helicase Linker (CHL) regulates RIG-I signaling In the autoinhibitory state of RIG-I, the helicase is in an open conformation and the CARDs are interacting with Hel2i. The missing CHL, in gray, passes near the RNA binding groove of the helicase domain as its spans the 40 Å distance between the C terminus of CARD2 and N terminus of Hel1. PAMP RNA and ATP binding induces a conformational change to close the helicase subdomains around the ligands and release the CHL and CARDs. Models were generated from crystal structures (PDB ID: 4A2W, left; composite model of two crystal structures PDB IDs: 4NQK and 5E3H, right and colored as in (B)). Domain schematic of RIG-I with the CHL sequence highlighted. Blue arrows, CARD2 residues known to contact the Hel2i domain in the CARD2:Hel2i interface in autoinhibited duck RIG-I. Negative and positive charged amino acids are labeled red and blue, respectively. Proline residues are in bold. IFN response of mock-transfected, WT and CHL RIG-I mutants measured by luciferase reporter assays in HEK293T cells under No RNA or 50 nM 5'ppp ds39 transfected conditions and reported as Relative Luciferase Units (RLU). Each condition was performed three times by two independent workers (n = 6), and individual trials are plotted. Bars represent mean value, and error bars reflect standard error. Dose-titration IFN response of mock-transfected, WT, and constitutively active CHL RIG-I mutants measured by luciferase reporter assays in HEK293T cells. Cells were transfected with either no RNA or 700 nM, 70 nM, or 7 nM of 5' ppp ds39 RNA. Each condition was performed two times by two independent workers (n = 4). Bars represent mean value, and error bars reflect standard error. 5'ppp ds39 stimulated IFN response of mock, WT, and constitutively active CHL RIG-I mutants. Each total IFN response in (D) was subtracted by each plasmid's no RNA transfection condition to show the amount of IFN response explicitly due to transfected RNA. Bars represent the average IFN response of each RNA condition minus the average no RNA IFN response, and error bars represent the average standard error of the two. IFN response assay of mock, WT, and Δ190–200 RIG-I as in (C) except the assay was performed in HEK293T RIG-I KO cells. Each condition was performed three times by two independent workers (n = 6), and individual trials are plotted. Bars represent mean value, and error bars reflect standard error. Download figure Download PowerPoint RIG-I must be activated by viral RNAs and not self RNAs that are abundant in the cytoplasm. Aberrant immune responses initiated by RIG-I are harmful and lead to autoinflammatory disorders (Roers et al, 2016; Crowl et al, 2017). RIG-I has evolved several mechanisms to discriminate self and non-self. PAMP RNAs are explicitly recognized by the CTD of RIG-I, which provides the first layer of RNA proofreading (Cui et al, 2008; Lu et al, 2010; Wang et al, 2010; Vela et al, 2012; Ramanathan et al, 2016). RIG-I then utilizes its ATPase activity to power translocation along the RNA, and dissociation from non-PAMP RNAs (Devarkar et al, 2018). However, to activate the signaling CARDs, the chosen RNA must be loaded into the helicase domain. Unlike CTD, which specifically recognizes blunt 5' tri- or diphosphorylated RNA ends, the helicase domain generally interacts with the phosphodiester backbone (Jiang et al, 2011; Kowalinski et al, 2011; Luo et al, 2011). Hence, regulatory mechanisms must exist to selectively load CTD-chosen RNAs into the helicase domain and filter out non-specific RNAs. However, we do not know how RNA binding into the helicase domain is regulated. Previous studies suggested that the CARD2:Hel2i interface plays a role in gating the helicase (Vela et al, 2012; Ramanathan et al, 2016), but the underlying mechanisms were not known. In this study, we have discovered a new regulatory region that controls RNA binding into the helicase domain. This finding was made serendipitously while studying the role of the ~56 aa linker that connects the CARDs to Hel1, referred to here as CHL (CARDs-Helicase Linker). No known functions were associated with the CHL, except that it tethers the CARDs to the helicase and that appropriately long CHL might be important for enabling intermolecular CARDs:CARDs interactions. Therefore, we were surprised to find that small deletions in CHL did not impair RIG-I signaling and instead constitutively activated RIG-I signaling without the addition of PAMP RNA. These results suggest that CHL is not a passive linker but a regulatory region that keeps RIG-I in the autoinhibitory state in the absence of PAMP RNA. To understand the role of CHL, we carried out a detailed mechanistic study of the CHL mutants using a combination of cellular, biochemical, and biophysical methods, including hydrogen–deuterium exchange and mass spectrometry (HDX-MS), equilibrium RNA binding, and stopped-flow kinetics. These studies revealed two essential roles of the CHL. One role of the CHL is to stabilize the CARD2:Hel2i interface and maintain RIG-I in an autoinhibitory conformation in the absence of PAMP RNA. The second role of CHL is to shield the helicase domain and antagonize non-specific RNAs. Both functions of the CHL depend on its negatively charged amino acids acting as an electrostatic gate. The RNA proofreading function of the CHL is greatly enhanced by a stable CARD2:Hel2i interface. The two work synergistically to minimize erroneous RNA binding events, ensuring that RNAs chosen by the CTD are specifically loaded into the helicase domain. The newly discovered regulatory functions of the CHL can be potentially leveraged in RIG-I-based therapeutics. Results CHL deletions constitutively activate RIG-I signaling in the absence of PAMP RNA The negatively charged CHL (186–241 aa) is predicted to be intrinsically disordered (Oates et al, 2013), and accordingly, not resolved in the autoinhibited duck RIG-I structure (Kowalinski et al, 2011) (Fig 1A). The AlphaFold structure also predicts that the CHL is disordered, demonstrated by a low confidence score and random coil conformation modeling (Jumper et al, 2021) (Fig EV1A and B). Additionally, the crystal structure of RIG-I suggests that the missing CHL is in close proximity to the helicase domain. Interestingly, AlphaFold structure prediction places the unstructured CHL within the RNA binding channel of the helicase domain (Fig EV1A). To investigate the role of CHL in RIG-I signaling, we made segmental deletions of 11 amino acids in the 190–240 region (Fig 1B) and tested the mutants in cell signaling assays. WT RIG-I and CHL mutants were transfected in HEK293T cells and their interferon response was measured using a dual-luciferase IFN-β promoter-reporter assay with and without PAMP RNA. As expected, in the absence of PAMP RNA, WT RIG-I showed minimal basal signaling response, but unexpectedly, three of the five CHL mutants showed close to 10-fold higher basal signaling response than WT RIG-I (Fig 1C). We verified that all the RIG-I proteins were expressed after transfection in the 293T cells (Fig EV1C). The CHL mutants that were constitutively activated had deleted regions close to CARD2 (Δ190–200, Δ200–210, and Δ210–220), whereas mutants with deletions close to Hel1 (Δ220–230 and Δ230–240) behaved like WT RIG-I and did not signal well in absence of PAMP RNA. The results indicate that the 190–220 region of the CHL is involved in autoinhibiting RIG-I signaling in the absence of PAMP RNA. The 220–240 region may have a different role. Click here to expand this figure. Figure EV1. Induction of the IFN response genes by the CHL mutants A. AlphaFold predicted structure of RIG-I shows CHL bound in the RNA binding pocket of the helicase domain. The colors correspond to model per-residue confidence (pLDDT): Dark blue, very high (pLDDT > 90), light blue, confident (90 > pLDDT > 70), yellow, low (70 > pLDDT > 50), orange, very low (pLDDT < 50). Regions below 50 pLDDT are predicted to be unstructured, like the CHL in orange. B. Graph demonstrating per-residue confidence (pLDDT) of AlphaFold prediction shown in (A), indicating low-confidence in the prediction of CHL structure. The CHL (186–241) is highlighted in orange. C. Western Blot confirms RIG-I expression in the reporter assays in Fig 1. In each experiment, pcDNA3.1 myc-tagged RIG-I constructs (approximately 108 kDa) were recognized with a primary α-Myc antibody. β-actin (approximately 42 kDa) was used as a normalization control. Numbers (left) refer to molecular weight in kDa. Note that HEK293T and HEK293T RIG-I KO Western blots were performed on separate gels. D–G. qRT–PCR assays show the induction of antiviral IFN response genes in the absence and presence of PAMP RNA, 5'ppp ds39. Note the Y-axis is in log scale. Each bar represents the mean ± SD. Each point represents a mechanical replicate (n = 2). H. Western blot to show induction of pIRF3 in RIG-I and RIG-I mutant transfected cells. Download figure Download PowerPoint When the cells were transfected with PAMP RNA, 5'ppp ds39, all tested RIG-I constructs responded with increased signaling (Fig 1C). To test the relative sensitivities of the wild-type RIG-I and the constitutively active CHL mutants to activation by the PAMP RNA, we titrated the cells with 7–700 nM PAMP RNA and measured the signaling responses (Fig 1D). The CHL mutants had an overall higher interferon signaling activity than wild-type RIG-I at all RNA concentrations. After subtracting the basal signaling response, we find that the PAMP signaling response of the CHL mutants at the lowest 7 nM PAMP RNA concentration is about 2-fold lower than wild-type RIG-I (1.7-fold decrease for WT, while CHL mutants decreased between 2.5-fold and 3.5-fold) (Fig 1E). The signaling response of the CHL mutants at intermediate or high RNA concentrations was comparable to wild-type RIG-I. Thus, CHL deletions only slightly impairs the RNA-dependent signaling activity relative to wild-type RIG-I at low PAMP RNA concentrations. To confirm that the signaling activity measured in HEK293T cells is not due to activation of endogenous RIG-I, we tested a minimal CHL mutant panel in HEK293T RIG-I KO cells (Fig 1F). Overall, we obtained the same results in RIG-I KO and 293T cells. The Δ190–200 RIG-I showed a 7-fold higher basal signaling activity in the RIG-I KO cells, which is similar to the 10-fold observed in the 293T cells. This confirms that the CHL mutants are constitutively activated in interferon signaling. To demonstrate that the constitutively active CHL mutant activates the antiviral interferon (IFN) response pathway, we measured the expression of several IFN response genes, including IFNB, MX1, ISG15, OAS1 using qRT–PCR in the absence and presence of 5'ppp ds39 PAMP RNA. The Δ190–200 RIG-I activated the IFN response genes and pIRF3 expression in the absence of PAMP RNA, whereas WT RIG-I required PAMP RNA for activation of these genes (Fig EV1D–H). These cellular studies demonstrate that CHL is a critical regulatory region that controls the RIG-I signaling pathway inducing the IFN response genes. CHL stabilizes the CARD2:Hel2i interface to minimize CARDs exposure and prevent non-specific RNA binding Small deletions in CHL can increase the basal signaling activity of RIG-I in two ways. First, the deletions may destabilize the autoinhibited conformation of RIG-I and spontaneously expose the CARDs independently of RNA binding. Additionally, the deletions may dysregulate RNA binding, promoting CARDs activation through promiscuous binding of self RNAs. To test the first possibility, we used differential HDX-MS analysis, a powerful technique to monitor CARDs exposure in RIG-I used previously (Zheng et al, 2015, 2018, 2019). The solvent exchange rates were significantly different between WT and Δ190–200 RIG-I in the absence of RNA. The CHL mutant showed a higher solvent exchange rate in many regions, including the CARDs (aa 1–186), Hel1 (aa 410–460), Hel2 (aa 695–756, 785–803), and Hel2i (aa 566–574 and 522–539) (Figs 2A and B, and EV2). This indicates that 190–200 deletion has a global effect on the autoinhibited conformation of RIG-I. In particular, increased solvent exchange at the CARD2:Hel2i interface, comprising the CARD2 latch peptide (aa 103–114) and the interacting Hel2i regions (aa 566–574 and 522–539) suggests this global effect is caused by the 190–200 deletion through destabilization of the CARD2:Hel2i interface. Figure 2. CHL stabilizes CARDs in the autoinhibitory conformation and inhibits stem RNA binding Hydrogen–Deuterium Exchange Mass Spectroscopy (HDX) heatmap of three comparisons: WT RIG-I vs Δ190–200 RIG-I without RNA; WT RIG-I with and without 5'-triphosphate (5'ppp) RNA, a RIG-I PAMP; and Δ190–200 RIG-I with and without 5'ppp RNA. The bar below indicates % deuteration of a given peptide region. White spaces indicate no sequence coverage, and grey represents regions in which the coverage was non-significant. Data from (A) modeled onto autoinhibited RIG-I (PDB ID: 4A2W) or activated RIG-I, which is a composite model of two crystal structures (PDB IDs: 5E3H and 4NQK). In the autoinhibited structure, only CARDs and Hel2i are colored according to the HDX results. RIG-I binding to ds26 stem RNA. The ATPase activity of WT RIG-I and Δ190–200 RIG-I was measured at increasing concentration of ds26 stem RNA (black: RNA, blue: DNA). The binding curves were fit using a hyperbola (Equation 5) to obtain the stem RNA KD,app for each RIG-I construct, as shown. The mean value is shown as a bar; error bars denote standard error. For each point, standard error was determined by the fit to each point's time course (mechanical replicates, n = 3). Model showing the consequences of CHL deletion. Small deletions in the CHL disrupt both the CHL positioning and the autoinhibitory CARD2:Hel2i interface, causing aberrant CARDs exposure and stem RNA binding. The coloring of the helicase subdomains, CTD, and CARDs is the same as in Fig 1. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Hydrogen–Deuterium Exchange Mass Spectroscopy (HDX) heatmap comparing WT RIG-I and Δ190–200 RIG-I The average ΔD2O% ± standard deviation between WT RIG-I and Δ190–200 RIG-I across all HDX time points. HDX Workbench colors each peptide according to the smooth color gradient HDX perturbation key shown in each indicated figure. Average ΔD2O% between −5% and 5% are considered non-significant and are colored gray. Download figure Download PowerPoint Next, we compared the solvent exchange rates in the absence and presence of 5'ppp ds10 hairpin, which stimulates CARDs release in WT RIG-I (Fig 2A and B, Appendix Figs S1 and S2). CARDs exposure was more prominent in WT RIG-I than in Δ190–200 RIG-I. This is understandable because the Δ190–200 RIG-I CARDs are already partially exposed before adding RNA. Differential HDX-MS also shows that RNA binding regions are protected in WT and Δ190–200 RIG-I to a similar extent. Hence, the CHL mutant is not defective in RNA binding, consistent with the increased signaling with PAMP RNA in the above assays. The most significant finding from the HDX-MS experiments is that CHL has a role in stabilizing RIG-I CARDs in the autoinhibitory conformation in the absence of RNA. To investigate if partial CARDs exposure dysregulates RNA binding, we measured the affinity of WT and Δ190–200 RIG-I for a stem RNA mimic. The ds26 stem was designed to contain a 17 bp dsRNA region flanked by 4 bp of dsDNA ends. The DNA ends block RIG-I from end binding, forcing it to bind internally in the RNA stem region, mimicking secondary stem structures in self RNAs. RNA KD,app values were determined from titration experiments monitoring RIG-I's RNA-dependent ATPase activity as a function of increasing RNA. As expected, WT RIG-I binds to ds26 stem with a very weak affinity and KD,app ~1250 nM (Fig 2C). Interestingly, Δ190–200 RIG-I showed a much higher affinity and KD,app of ~20 nM, which is 50-fold tighter than WT RIG-I. As shown (Fig 1D), it appears that a small deletion in CHL in the 190–220 region exposes the CARDs and disrupts the positioning of CHL to enable stem RNA to bind into the helicase domain. These results indicate that the hyperactive signaling response of the CHL mutants could be both due to partially exposed CARDs and promiscuous binding and activation by self RNAs in the cytoplasm. Thus, CHL has a dual role—stabilize the CARDs in the autoinhibitory conformation and regulate RNA binding into the helicase domain. The negative charges in the CHL are essential in autoinhibiting aberrant RIG-I signaling How does the deletion of 11 aa in CHL destabilize the CARD2:Hel2i interface? Is it a length effect, or does the deletion disrupt some autoinhibitory interactions of the CHL with the helicase? An unstructured CHL of 56 aa should be theoretically 168 Å long (assuming ~3 Å per aa), which is four times longer than the distance it needs to span between CARD2 and Hel1 (~40 Å). A 33 Å deletion is not long enough to shorten the linker and disrupt the interface if the CHL is truly unstructured, as the Alphfold structure prediction indicates (Fig EV1A and B). Thus, CHL may have specific, dynamic interactions with the helicase domain that stabilizes the autoinhibitory CARDs conformation. Sequence analysis of several RIG-I homologs from bony fish to mammals shows that CHL is highly negatively charged. Although the CHL sequence conservation is poor relative to the helicase domain, the negative charges in CHL are well conserved (Fig 3A). Between residues, 186–220, 11 of the 35 amino acids of the human CHL are either aspartate or glutamate, with only three lysine
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