Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are cytosolic innate immune sensors that recognize viral double-stranded (ds)RNAs. RLRs (RIG-I, MDA5 and LGP2) are members of the so-called Superfamily II (SF2) helicases/translocases. They share a multi-domain architecture that consists of a central SF2 ATPase domain accompanied by two N-terminal tandem caspase activation and recruitment domains (2CARD, only in RIG-I and MDA5) and a C-terminal regulatory domain (CTD or RD) (Rawling and Pyle, 2014). The SF2 domain itself is built out of an N-terminal RecA-like domain (1A) and a C-terminal RecA-like domain (2A) that together form an ATP-binding pocket, as well as an insertion domain (domain 2B). SF2 and RD are crucial for RNA-recognition of RLRs, whereas 2CARD communicates successful RNA-binding events to downstream signaling factors (Jiang et al., 2011; Kowalinski et al., 2011; Luo et al., 2011). Specifically, RIG-I detects dsRNA ends harbouring 5’ tri- or diphosphates (Goubau et al., 2014; Schlee et al., 2009; Schmidt et al., 2009). Simultaneous binding of an RNA ligand and ATP to RIG-I switches the protein into an active state in which the otherwise shielded 2CARD is released (Zheng et al., 2015). Activated RIG-I homo-tetramerizes via 2CARD (Jiang et al., 2012) and nucleates the polymerisation of its adapter protein, mitochondrial antiviral-signaling (MAVS), to elicit the innate immune signaling cascade (Peisley et al., 2014; Wu et al., 2014).

The similarity of epitopes of viral RNA recognized by RLRs, in particular dsRNA stems, to some endogenous ribonucleic acids has required the immune system to develop mechanisms besides merely recognizing 5’-di/triphosphate-containing RNA ends to discriminate self from non-self. Self-ribonucleic acids for instance are shielded from RLRs by introducing 2’O-methylations or by destabilizing double-stranded parts through A-to-I editing (Chung et al., 2018; Devarkar et al., 2016; Liddicoat et al., 2015; Schuberth-Wagner et al., 2015). In addition, we and others have been able to show that the SF2 domain of RLRs itself confers a proof-reading activity by removing RIG-I from self-RNA so as to avoid autoimmunity (Anchisi et al., 2015; Lässig et al., 2015; Louber et al., 2015; Rawling et al., 2015). In particular, ATP turnover can lead to translocation on dsRNA stems that could remove the protein from the RNA and reinstall the inactivated state (Myong et al., 2009; Yao et al., 2015).

Deficiencies in any of these mechanisms can lead to immune recognition of self-RNAs (Ahmad et al., 2018; Chiang et al., 2018). For instance, single-nucleotide polymorphisms (SNPs) in RLR genes are known to cause system-wide autoimmune diseases such as Aicardi-Goutière syndrome (AGS) (Oda et al., 2014; Rice et al., 2014), Singleton-Merten syndrome (SMS) (Jang et al., 2015; Rutsch et al., 2015), systemic lupus erythematosus (SLE) (Cunninghame Graham et al., 2011; Pettersson et al., 2017; Van Eyck et al., 2015) or type 1 diabetes (Liu et al., 2009; Smyth et al., 2006). The molecular basis for the development of these diseases is in many cases not understood, because structural data on these RLR variants have been missing.

In this work, we present the biochemical and structural analysis of the RIG-I SMS variant C268F and unveil an ATP-independent signaling mechanism. We show that active site rearrangements of several amino acid side chains in RIG-I C268F mimic an ATP-bound state and activate the protein for signaling upon recognition of RNA ligands in the absence of ATP.

The RIG-I SMS variants C268F and E373A are located within SF2 ATP binding and hydrolysis motifs I and II, respectively, (Fairman-Williams et al., 2010) (Figure 1—figure supplement 1) and were previously shown to be constitutively active in reporter cells without any external dsRNA trigger (Jang et al., 2015; Lässig et al., 2015) (Figure 1—figure supplement 2A). RIG-I E373A’s enhanced immune signaling ability can be explained by proficient ATP binding but reduced ATP hydrolysis, since the motif II glutamate is implicated in positioning and polarizing the attacking water molecule in the hydrolysis reaction. The stabilized ATP state leads to increased binding of and activation by endogenous dsRNA (Lässig et al., 2015; Louber et al., 2015). By contrast, the molecular basis for activation of RIG-I C268F is still unknown as this mutation is located in motif I, which is normally associated with ATP binding and which is critical for RIG-I activation. For instance, a designed and widely used mutant with a defect in the invariant ATP phosphate-binding motif I lysine, K270A, possesses reduced ATP binding properties (Rawling et al., 2015) and the RIG-I K270A/I mutant is deficient in inducing an immune response (Lässig et al., 2015; Yoneyama et al., 2005). By contrast, the RIG-I SMS variant C268F shows the opposite effect and is constitutively active, although this mutation is only two amino acids away from K270A in the same ATP phosphate-binding ‘P-loop’ in motif I (Figure 1—figure supplement 2A).

To analyze whether the autoimmune activity of the SMS variant RIG-I C268F is RNA-dependent, we designed a double-mutant that is defective in RNA-binding and carried out interferon (IFN)-β-promoter-driven luciferase reporter assays with overexpressed proteins in HEK293T RIG-I KO cells (Figure 1A). In particular, we used a previously described T347A point mutation in the RNA-binding interface of the N-terminal RecA-like domain (1A) (Figure 1—figure supplement 1), that abrogates the signaling of wild type (wt) RIG-I in infected cells and decreases the affinity for dsRNA in vitro (Lässig et al., 2015). Our assays show, that RIG-I C268F, T347A fails to induce the IFN-β promoter in uninfected as well as in 19mer 5’-triphosphate (ppp)-dsRNA-stimulated cells, indicating that the RIG-I SMS single-mutant C268F induces immune signaling only if bound to endogenous or transfected RNA. In addition, competition assays of RIG-I C268F titrated with signaling-deficient RIG-I lacking the 2CARD domain (RIG-I Δ2CARD) or with RIG-I Δ2CARD E373Q also gradually decreased the IFN-β promoter-driven luciferase activity (Figure 1—figure supplement 2B). Thus, RIG-I C268F as well as RIG-I Δ2CARD (E373Q) seem to recognize identical RNA substrates which are, however, saturated with signaling-deficient RIG-I Δ2CARD (E373Q) at higher transfected DNA concentrations thus preventing an immune response. Furthermore, we can exclude the possibility that the RIG-I C268F SMS mutation leads to a liberation of the 2CARD signaling module in the absence of RNA, for example by unfolding SF2. Instead, signaling by the RIG-I C268F SMS variant is dependent on 2CARD release triggered by binding to RNA molecules, as it is in wild type RIG-I.

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To validate the intact RNA-binding properties of RIG-I C268F, we performed in vitro fluorescence anisotropy assays of purified proteins with a labeled hairpin (hp) RNA containing non-base-paired RNA ends (Figure 1B). We chose to use a double-stranded RNA ligand without blunt ends to suppress the dominant binding of RIG-I’s RD to terminal RNA base pairs and to simulate recognition of endogenous-like RNA species that could be present within the cytosol. As expected, wild type RIG-I shows a moderate binding affinity to this ligand that is further decreased in the presence of ATP. By contrast, RIG-I C268F displays an already increased affinity to the hpRNA that is even enhanced in the presence of ATP. This confirms the previous results for RIG-I C268F showing an increased co-purification with endogenous RNA molecules (Lässig et al., 2015) and indicates that RIG-I C268F has two conformations (apo and ATP-bound) that have increased dsRNA affinity.

Since an intact SF2 ATPase domain of RIG-I is needed so that signaling only occurs when foreign RNA molecules are recognized (Louber et al., 2015; Rawling et al., 2015), we further analyzed the ATP binding and hydrolysis properties of RIG-I C268F in vitro. The ATP-binding-deficient and hydrolysis-deficient motif I mutant RIG-I K270I and the hydrolysis-deficient motif II mutant RIG-I E373Q both served as references. ATP hydrolysis assays with ³²P-labeled ATP confirmed a loss of catalytic activity of RIG-I C268F and of both motif I and II mutants in the presence of dsRNA (Figure 2A). In accordance with our RNA-binding experiments, this evidence supports a model in which RIG-I C268F has defects in dissociating from endogenous RNA, because it lacks the capability for ATP turnover and hence translocation. To further analyze the ATP-binding properties of the RIG-I SMS variant, we conducted a tryptophan fluorescence-based FRET assay with MANT-ATP (Rawling et al., 2015) (Figure 2B). In the absence of any RNA ligand, both wild type RIG-I and ATP-hydrolysis-deficient RIG-I E373Q show comparable affinities for MANT-ATP and MANT-ATPγS in the low µM range (Table 1). As expected, ATP-binding-deficient RIG-I K270I has a reduced affinity for ATP (in the medium µM range). In accordance with previous data (Kohlway et al., 2013), the presence of RNA increases the affinity of wtRIG-I and RIG-I E373Q, but not of RIG-I K270I for MANT-ATP or MANT-ATPγS. Interestingly, like RIG-I K270I, the RIG-I SMS variant C268F displays reduced ATP-binding affinities compared to that of wtRIG-I or RIG-I E373Q independently of the availability of an RNA ligand (Figure 2B, Table 1). This is puzzling as both RNA and ATP binding are normally needed to induce a molecular switch within RIG-I in order to release the 2CARD module and to activate an immune response (Shah et al., 2018; Zheng et al., 2015). RIG-I C268F might thus be able to signal even in the absence of a bound ATP molecule. However, even though it is possible that under cellular conditions of 1 mM ATP or even higher, ATP is still bound by both motif I mutants, the molecular switch to release 2CARD is only triggered in RIG-I C268F but not in RIG-I K270I (Figure 1—figure supplement 2A). These peculiarities of the ATP-bound states of the motif I mutants need to be addressed in future studies.

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To further investigate the influence of ATP binding to RIG-I C268F, we mutated two residues that are implicated in ATP adenine recognition, R244A and Q247A (Figure 2—figure supplement 1), thereby further lowering the ATP-binding affinity of the SMS mutant, and tested the ability of the resulting triple-mutant to induce an immune response (Figure 2C). We expected to see no decrease in the autoimmune signaling activity of the triple-mutant compared to that of the RIG-I SMS single-mutant if the single mutant was not able to bind ATP under cellular conditions. However, compared to the SMS variant itself, the IFN-β promoter activity of the ATP-binding mutant RIG-I R244A, Q247A, C268F was reduced, albeit still significantly higher than that of wild type RIG-I. Hence, these data support a model in which RIG-I C268F is able to release its 2CARD domain and start a signaling cascade by a process that is at least partly independent of ATP.

In order to elucidate the molecular basis of autoimmune signaling by RIG-I C268F, we went on to crystalize RIG-I Δ2CARD C268F in the presence of a 14mer dsRNA and ADP·BeF_x or ATP and determined the corresponding structures (Figure 3—figure supplement 1A). Intriguingly, despite an overall high structural similarity to wtRIG-I Δ2CARD bound to dsRNA (Jiang et al., 2011), the SMS mutant shows crucial amino acid side chain rearrangements in the active site that stabilize the protein in an activated state but prevent binding and co-crystalization of a nucleotide (Figure 3A, Figure 3—figure supplement 1B). In particular, the bulky F268 side chain displaces the evolutionary invariant motif I K270 from its central ATP phosphate-binding position in the P-loop, into a position where the Nε of K270 is situated at a site normally occupied by the Mg²⁺ ion (Video 1). As a result, K270 now forms a salt bridge with E702 from the C-terminal RecA-like domain 2A, which in turn occupies the ATP γ-phosphate binding site. The resulting overall conformation resembles the ATP-bound state of RIG-I, but without ATP, and could thus explain how C268F is able to signal independently of any nucleotide. In order to clarify the impact of the salt bridge, we mutated both side chains in RIG-I C268F and analyzed the resulting double-mutants in our IFN-β promoter activity assay (Figure 3B). We expected that a disturbance of the salt-bridge formation in RIG-I C268F would lead to a loss of autoimmune signaling. Indeed, mutation of K270 in RecA-like domain A1 renders RIG-I C268F inactive, probably as the result of a mixture of (i) prevention of the activation of RIG-I C268F in the absence of ATP by disrupting the salt bridge, and/or (ii) the failure to bind ATP altogether through impaired ATP phosphate coordination and thus an impaired 2CARD release. In contrast to our ATP-binding triple mutant R244A, Q247A, C268F, which still allows formation of the salt bridge in the absence of ATP, mutation of motif I K270 in RIG-I C268F thus renders the protein inactive. Mutation of E702 in the RecA-like domain 2A of RIG-I C268F, by contrast, does not disrupt constitutive signaling of the protein, although this activity is at a reduced level compared to that of RIG-I C268F. An explanation for this might be E702's localization within the SF2 helicase motif V, which couples RNA-binding-induced ATP hydrolysis with movement on dsRNA. Similar to our previously described V699A mutant in the same motif (Lässig et al., 2015), E702A alone already induces an autoimmune phenotype similar to that induced by RIG-I C268F, E702A. The most plausible explanation for these data is that RIG-I C268F stabilizes an ATP-like state in the absence of ATP, but still allows formation of a proper ATP-bound state. Although E702A could reduce the former (by disrupting the salt bridge), it might stabilize the latter by sterically allowing ATP binding or by increasing the interaction with RNA.

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Video 1

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In summary, our data suggest that the RIG-I C268F SMS mutation stabilizes the signal-on state of RIG-I in the presence of RNA but absence of ATP through a salt bridge between K270 from domain 1A and E702 from 2A. Unlike wild type RIG-I, which requires both RNA and ATP bound in order to be activated for downstream signaling, RIG-I C268F can signal independently of ATP (Figure 2C) in the presence of RNA (Figure 1A). However, our data also show that ATP further increases the binding of RIG-I C268F to internal dsRNA stems (Figure 1B) and that the engineering of ATP-binding mutations R244A, Q247A into RIG-I C268F at least partly reduces its signaling activity in response to endogenous or ppp-dsRNA RNA (Figure 2C). Even though we were not able to co-crystalize RIG-I C268F in the presence of a nucleotide, it is very possible that binding of ATP at high molecular concentrations, such as those present in a cellular context, further contributes to the pathogenic signaling activity of the protein. In principle, ATP binding would be sterically allowed if the salt-bridge-forming residue E702 occupies a site as in wtRIG-I. It is not yet clear what happens at the Mg²⁺-binding site, as sterically F268 would not allow a canonical positioning of motif I K270 and might thus prevent binding of Mg²⁺. Perhaps K270 remains at the displaced site even in the presence of ATP and simply ‘mimics’ Mg²⁺. Such a scenario could still lead to reduced binding of ATP but would prevent ATP hydrolysis, as observed, because of a substantially altered charge distribution. This mechanism unifies the molecular basis for the development of SMS by both RIG-I variants (Figure 4). Unlike wtRIG-I, which consumes ATP and uses ATP turnover to decrease its affinity to self-RNA (Figure 4A), both motif I and II SMS mutations either mimic or freeze RIG-I in an ATP-bound state (Figure 4B). As a consequence, the inability to hydrolyze ATP and thus to dissociate actively from RNA (Figure 1B, Figure 2A) traps the protein in an activated state and thus explains autoimmune signaling in response to self-RNA.

Figure 4

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In conclusion, we provide for the first time a detailed biochemical and structural analysis of an RLR autoimmune disease variant. Slight intramolecular rearrangements within the RIG-I C268F ATP-binding pocket appear to compensate for ATP binding and render the protein active in the presence of dsRNA only. At the same time, loss of proof-reading activity leads to increased activation by endogenous RNA. This unusual gain-of-function mutation reveals, for the first time to our knowledge, that a P-loop mutation can mimic the effects of ATP binding.

Diffraction data have been deposited in PDB under the accession code 6GPG.

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Author details

1. Charlotte Lässig

   1. Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany
   2. Gene Center, Ludwig-Maximilians-Universität München, Munich, Germany

   Contribution

   Conceptualization, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6253-7880

2. Katja Lammens

   1. Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany
   2. Gene Center, Ludwig-Maximilians-Universität München, Munich, Germany

   Contribution

   Formal analysis, Supervision, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4438-1381

3. Jacob Lucián Gorenflos López

   1. Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany
   2. Gene Center, Ludwig-Maximilians-Universität München, Munich, Germany

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

4. Sebastian Michalski

   1. Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany
   2. Gene Center, Ludwig-Maximilians-Universität München, Munich, Germany

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

5. Olga Fettscher

   1. Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany
   2. Gene Center, Ludwig-Maximilians-Universität München, Munich, Germany

   Contribution

   Investigation, Cloned and tested mutant proteins for cell-based assays

   Competing interests

   No competing interests declared

6. Karl-Peter Hopfner

   1. Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany
   2. Gene Center, Ludwig-Maximilians-Universität München, Munich, Germany
   3. Center for Integrated Protein Science Munich, Munich, Germany

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   hopfner@genzentrum.lmu.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4528-8357

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