Structure

Short Article

Visualizing the Determinants of Viral RNA Recognition by Innate Immune Sensor RIG-I Dahai Luo,1,4 Andrew Kohlway,2 Adriana Vela,2 and Anna Marie Pyle1,3,4,* 1Department of Molecular, Cellular, and Developmental Biology 2Department of Molecular Biophysics and Biochemistry 3Department of Chemistry Yale University, New Haven, CT 06520, USA 4Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA

*Correspondence: anna.pyle@yale.edu

http://dx.doi.org/10.1016/j.str.2012.08.029

SUMMARY

Retinoic acid inducible gene-I (RIG-I) is a key intra- cellular immune receptor for pathogenic RNAs, particularly from RNA viruses. Here, we report the crystal structure of human RIG-I bound to a 50

triphosphorylated RNA hairpin and ADP nucleotide at 2.8 Å resolution. The RNA ligand contains all structural features that are essential for optimal recognition by RIG-I, as it mimics the panhandle- like signatures within the genome of negative- stranded RNA viruses. RIG-I adopts an intermediate, semiclosed conformation in this product state of ATP hydrolysis. The structure of this complex allows us to visualize the first steps in RIG-I recognition and acti- vation upon viral infection.

INTRODUCTION

Pathogen recognition receptors (PRRs) are signaling proteins

that continually survey cells for the presence of pathogen associ-

ated molecular patterns (PAMPs). Retinoic acid inducible gene I

(RIG-I) is a major cellular PRR that senses viral RNA PAMPs in

the cytoplasm of infected cells (Kato et al., 2011; Yoneyama

et al., 2004). RIG-I recognizes a broad spectrum of viruses,

including the negative-stranded vesicular stomatitis virus, influ-

enza, and rabies viruses, and also positive-stranded viruses

such as dengue and hepatitis C virus (Kawai and Akira, 2007;

Ramos and Gale, 2011). Defective viral replication by Sendai

virus and influenza virus generates short subgenomic RNAs

that may be a principal ligand for RIG-I during viral infection

(BaumandGarcı́a-Sastre, 2011;Baumet al., 2011). At themolec-

ular level, RIG-I preferentially recognizes double stranded RNAs

that contain a triphosphate moiety at the 50 end, exemplified by thepanhandle-likeRNAsof negative-strand viruses such as influ-

enza (Hornung et al., 2006; Pichlmair et al., 2006; Schlee et al.,

2009). Recent biochemical and structural studies have shown

that the C-terminal domain (CTD) of RIG-I recognizes duplex

termini, interacting specifically with terminal 50 triphosphate moieties (Cui et al., 2008; Lu et al., 2010; Wang et al., 2010).

Structure 20, 1983–19

The central SF2 helicase domain (HEL) binds internally to the

double-stranded RNA (dsRNA) backbone (Jiang et al., 2011; Ko-

walinski et al., 2011; Luo et al., 2011). A pincer domain connects

the CTD and the HEL domains and provides mechanical support

for coordinated RNA recognition by the two domains (Luo et al.,

2011). TheN terminal tandemcaspase activation and recruitment

domains (CARDs) are responsible for downstream signaling,

leading to the expression of antiviral interferon-stimulated genes

(Jiang and Chen, 2011; Ramos and Gale, 2011).

The current model of RIG-I activation suggests that the

binding ofRNAby theHELandCTDgenerates a nanomechanical

force that releases an inhibitory conformation imposed by the

CARD domains, a process that also requires ATPase activity

through an unknown mechanism (Kowalinski et al., 2011; Luo

et al., 2011). Identifying the molecular determinants for RNA

recognition and understanding how RIG-I distinguishes viral

RNA from cellular RNA represent important unanswered ques-

tions in the field of innate immunity. Here, we report the crystal

structure of RIG-I in complex with a 50 triphosphorylated double-stranded RNA and adenosine nucleotide, thereby

providing the biologically relevant snapshot of viral PAMP recog-

nition by RIG-I. We show that binding of different ATP analogs

induces specific conformational changes within the protein,

verifying the structural observations and supporting a tightly

regulated, multistep activation mechanism of RIG-I.

RESULTS AND DISCUSSION

To unravel the molecular details of viral PAMP recognition by

RIG-I, we designed a hairpin RNA (hereafter named as 50

ppp8L which contains a 50 triphosphate moiety and a stem of 8 base pairs that is terminated by a UUCG tetra loop) that mimics

the panhandle-like genome of negative-stranded RNA viruses

(Figures S1 and S2 available online). We cocrystallized 50

ppp8L with a human RIG-I construct that lacks the CARD

domains (RIG-I [DCARDs: 1–238]; Figure 1). All atoms of the

RNA hairpin are observed and unambiguously built into the

2.8 Å density map (Figure 1C; Table 1).

The overall structure of the complex (RIG-I (DCARDs: 1–238):

50 ppp8L: ADP-Mg2+) is similar to the RIG-I:dsRNA10 structure reported previously (rmsd = 0.38 Å for 559 superimposed Ca

atoms) (Luo et al., 2011). However, in the structure reported

88, November 7, 2012 ª2012 Elsevier Ltd All rights reserved 1983

Figure 1. Ternary Complex of RIG-I

(DCARDs 1–238): 50 ppp8L: ADP-Mg2+

(A) Structure of the 50 triphosphorylated hairpin RNA (50 ppp8L, in purple with 50 GTP in red) bound at the center of the RIG-I (DCARDs). Bound ADP-

Mg2+ is in purple.

(B) The 50 triphosphate binding site at CTD. Fo-Fc omit map is in green and contoured at 3.5 s.

(C) Superposition of RIG-I with 50 triphosphory- lated hairpin RNA and RIG-I with 50 hydroxyl dsRNA in gray (PDB: 2ykg).

See also Figures S1 and S2.

Structure

Structure of RIG-I, 50 ppp-dsRNA, and ADP

here, the CTD encapsulates the 50 triphosphate moiety at the duplex terminus. Functional groups along the RNA duplex

interact with the HEL1 and HEL2i domains as observed

previously. Importantly, one can now observe the position of

bound nucleotide, revealing that ADP interacts exclusively

with conserved ATPase motifs localized in HEL1 (Figure 1A).

HEL2 is not involved in RNA binding or ADP binding (Figure 1).

The protein conformation observed in this structure is likely to

be biologically relevant because we observe that 50 ppp8L RNA readily stimulates efficient ATP hydrolysis by RIG-I (Fig-

ure S3; Table S1).

The RNA triphosphate is specifically recognized by the RIG-I

CTD, which forms a network of electrostatic and hydrophobic

interactions (Figures 1B and 1C). Specifically, the a-phosphate

interacts with K861 and K888 and the b-phosphate interacts

with H847 and K858. Intriguingly, the g phosphate (for which

there is strong electron density) does not form any direct

contacts with the protein in this structure, suggesting that it is

not a major recognition determinant. If the triphosphate moiety

were to adopt a more extended configuration in an alternative

conformational state, the g phosphate would be likely to estab-

lish interactions with the K849 and K851 residues, as hypothe-

sized in structural studies of the isolated CTD in complex with

triphosphorylated RNA (Figure S1B) (Lu et al., 2010; Wang

et al., 2010). The structure of the intact complex (RIG-I (DCARDs:

1–238): 50 ppp8L: ADP-Mg2+) indicates that the a and b phos- phates at the 50 RNA terminus are particularly critical for RIG-I

1984 Structure 20, 1983–1988, November 7, 2012 ª2012 Elsevier Ltd All rights reserved

recognition. This may be due to the fact

that RNA g phosphates in the cell are

often hydrolyzed by host and viral RNA

triphosphatases (Decroly et al., 2012),

perhaps necessitating that RIG-I evolve

primary binding to a 50 diphosphate. Interactions involving the b phosphate

appear to be particularly important, as

they have global consequences for the

structure of the complex. Specifically,

contacts with H847 and K858 rigidify

the intervening loop and deliver it to

the blunt end of the triphosphorylated

RNA, enabling aromatic loop residue

F853 to stack on the first base pair of

the duplex and form energetically

favorable p-p interactions (Figure 1C).

Mutations that disrupt this interdigitated

network of contacts weaken triphosphorylated RNA binding

by RIG-I (Figure S4) (Wang et al., 2010). Together, they help

RIG-I to select the correct pathogenic RNA from the vast pool

of capped cellular RNAs.

Backbone atoms of the RNA duplex form an extensive set of

interactions with the HEL2i domain, providing further insights

into the mechanism of duplex recognition by RLR proteins. The

shape-selective RNA interface explains why RIG-I is capable of

binding to double-stranded RNAs from diverse viruses (Fig-

ure 1A; Figure S2). Significantly, the UUCG tetraloop at the

hairpin terminus is absorbed into an RNA binding tunnel and

does not establish any base specific contacts with RIG-I. The

structure demonstrates that a variety of RNA motifs, including

mismatches and ordered loops, would be readily accommo-

dated at the ‘‘far end’’ of the RIG-I RNA binding tunnel (i.e., the

end opposite 50 ppp binding). This is likely to be particularly important for RIG-I detection of negative-sense viral genomic

RNAs, including influenza, rabies, parainfluenza, and respiratory

syncytial virus, which also form short terminal duplexes capped

by loops (Figure S2).

In addition to RNA recognition, the structure of the complex

(which contains ADP-Mg2+) provides additional insights into

RIG-I recognition of bound nucleotide. The phosphates of ADP

interact with K270 and T271 (motif I) and with D372 (motif II)

through a bridging Mg2+ (Figure 2A). The adenine nucleobase

is recognized by Q247 (Q motif) and stacks between R244 and

F241. A comparison of available RIG-I:nucleotide structures

Table 1. Crystallographic Statistics

Data Collection

Structure RIG-I (DCARDs 1–238): 50 ppp8L: ADP-Mg2+

Space group P212121

Cell dimensions (Å) 47.7, 76.2, 221.2

Resolution (Å) 47.7–2.8 (2.95–2.8)a

R merge (%) 13.2 (61.4)

I/s 12.7 (3.7)

Completeness (%) 98.5 (98.7)

Redundancy 3.8 (3.9)

Refinement

Resolution (Å) 24.9–2.8

R work / R free (%) 21.8/28.6

No. atoms 5,542

Macromolecules 5,411

Ligands 61

Water 70

B factors (Å2) 54.2

Macromolecules 54.4

Solvent 35.2

Ramachandran analysis

Favored (%) 93

Additionally allowed (%) 6.2

Not favored (%) 0.8

Rmsd

Bond lengths (Å) 0.008

Bond angles (�) 1.15 aHighest resolution shell is shown in parentheses.

Structure

Structure of RIG-I, 50 ppp-dsRNA, and ADP

reveals that RIG-I (and perhaps related RLRs and DEAD-box

proteins) has a distinctive strategy for binding and activating

nucleotide ligands. Similar to DEAD box proteins, the helicase

domain of RIG-I is in an open conformation in the absence of

RNA substrate (Kowalinski et al., 2011; Luo et al., 2011; Pyle,

2008). In the presence of RNA and the ATP analog ADP-AlF3,

the helicase domain adopts the closed conformation, bringing

motifs I and VI into proximity 14. In complex with ADP-Mg2+, as

observed here, RIG-I adopts an intermediate, semiclosed state

that lacks contacts with motif VI from HEL2 (Figure 2B). Interest-

ingly, a similar semiclosed conformation was reported in the

structure of RIG-I with RNA and ADP-BeF3 (Figure 2C) (Jiang

et al., 2011), which may represent a transient state prior to

a completely closed ATP-bound state. Taken together, these

structures show that a bona fide closed conformation of the

helicase core is only captured in the presence of both dsRNA

and ADP-AlF3 and in the absence of CTD, indicating that RIG-I

conformation is exceptionally sensitive to ATP binding, hydro-

lysis, and product release. Importantly, the process of ATP

hydrolysis moves the CTD and HEL2i in opposite directions (Fig-

ure 3; Movie S1), which likely allows the CARDs to be released

from HEL2i (Kowalinski et al., 2011; Luo et al., 2011). This

provides a striking example of the conversion of chemical energy

into mechanical force and activation of a signaling relay.

Structure 20, 1983–19

To examine these nucleotide-dependent conformational

changes in solution, we performed a hydrodynamic analysis of

the RIG-I-RNA complex using sedimentation velocity analytical

ultracentrifugation. We observe a large shift in the sedimenta-

tion coefficient upon ADP-AlFx binding to the complex

(6.9% change in peak S value, Figure 2D). By contrast, binding

of ADP-BeF3 or ADP increases the peak S value only 4%

and 2% relative to the nucleotide-free state, respectively (Fig-

ure 2D). An increase in S value indicates compaction of the

hydrodynamic radius of the complex, and this correlates well

with available structural data (Jiang et al., 2011; Kowalinski

et al., 2011; Luo et al., 2011), as the greatest structural compac-

tion is observed in the presence of ADP-AlF3 (Figure 2D,

data shown for the full-length RIG-I). We suggest that ADP-

AlFx mimics the transition state of ATP hydrolysis, while

ADP-BeF3 likely mimics the initial ATP binding to the RecA-

like HEL1 domain. ADP is obviously the product bound state

during the ATP hydrolysis cycle of RIG-I. Importantly, we do

not observe functional interactions between RIG-I protein

molecules in the presence or absence of RNA. RIG-I and its

coupling cycle are therefore likely to be different from the

homologous MDA5, which cooperatively binds RNA (Berke

and Modis, 2012; Peisley et al., 2011).

In conclusion, it is now possible to visualize the conformational

response of RIG-I to binding of its two ligands, triphosphorylated

duplex RNA and nucleotide, and to envision the resultant

influence on antiviral signaling. While intriguing in their dynamic

implications, these snapshots also provide vital information

for the rational design of therapeutics that modulates RIG-I-

mediated immune responses.

EXPERIMENTAL PROCEDURES

Cloning, Expression, and Purification

The full-length RIG-I and N-terminal CARDs (1–238) deletion constructs,

hereafter named RIG-I (DCARDs 1–238), was cloned into the pET-SUMO

vector (Invitrogen). Transformed Rosetta II (DE3) Escherichia coli cells (Nova-

gen) were grown at 37�C in Luria broth medium supplemented with 40 mgml�1

kanamycin and 34 mg ml�1 chloramphenicol to an OD600nm of 0.6–0.8. Protein expression was induced at 18�C by adding isopropyl-b-D-thiogalactopyrano- side (IPTG) to a final concentration of 0.5 mM. After 20 hr growth, cells were-

harvested by centrifugation at 8,0003 g for 10min at 4�Cand stored at�20�C. Cells resuspended in buffer A (25 mM HEPES [pH 8.0], 0.5 M NaCl, 10 mM

imidazole, 10% glycerol, 5 mM b-ME) were lysed by passing three times

through a MicroFluidizer at 15,000 psi and the lysate was clarified by centrifu-

gation at 15,000 3 g for 60 min at 4�C. The supernatant was purified by batch binding with QIAGEN Ni-NTA beads. The beads were collected in Biorad

polyprep columns and the SUMO-tagged proteins were eluted with buffer B

(25 mM HEPES [pH 8.0], 0.3 M NaCl, 10% glycerol, 5 mM b-ME, 200 mM

imidazole). The fraction containing His6-Sumo-RIG-I was then digested with

ulp protease (Invitrogen), 4�C overnight. The cleavage mixture was loaded onto a HisTrap HP column to remove the His6-Sumo protein and ulp protease

from the mixture. The recombinant protein was then further purified by using

a HiTrap Heparin HP column (GE Healthcare) by running buffer C with an

additional 1 M NaCl gradient. Concentrated proteins were subjected to a final

gel-filtration purification step through a HiPrep 16/60 Superdex 200 column

(Amersham Bioscience) in buffer D (25 mM HEPES [pH 7.4], 150 mM NaCl,

2 mM MgCl2, 5% glycerol, 5 mM b-ME). Fractions containing monomeric

RIG-I were pooled, concentrated, and stored at �80�C. Recombinant protein RIG-I (DCARDs: 1–238) was expressed and purified using the same method.

The concentrations of the proteins were determined by measuring the

absorbance at 280 nm by using extinction coefficients of 95,300 M�1 cm�1

for full-length RIG-I and 60,040 M�1 cm�1 for RIG-I (DCARDs: 1–238).

88, November 7, 2012 ª2012 Elsevier Ltd All rights reserved 1985

Figure 2. ATP Binding and Hydrolysis by

RIG-I

(A) Interactions between human RIG-I and

ADP-Mg2+.

(B) Duck RIG-I with ADP-AlF3-Mg2+ (PDB: 4A36).

(C) Human RIG-I with ADP-BeF3-Mg2+ (PDB:

3TMI).

(D) Hydrodynamic analysis using sedimentation

velocity. Shown are the calculated distribution c(s)

versus s20,w of RIG-I:fUA10, RIG-I:fUA10:ADP-

AlFx (red), RIG-I:fUA10: ADP-BeF3 (blue), RIG-

I:fUA10: ADP (green). The peak values for the c(S)

distributions are 5.49S, 5.87S, 5.71S, and 5.60S,

which correspond to frictional coefficients of 1.58,

1.47, 1.51, and 1.54, respectively.

See also Figure S3 and Table S1.

Structure

Structure of RIG-I, 50 ppp-dsRNA, and ADP

RNA Preparation

The 50 triphosphorylated RNA hairpin (hereafter named 50 ppp8L) was produced by in vitro transcription using a synthetic dsDNA template (top

strand: 50-GTAATACGACTCACTATA GG CGCGGC ttcg GCCGCG CC-30) and purified by gel extraction (20% PAGE with 8 M urea).

Crystallization and Data Collection

To grow the crystals of the ternary complex of RIG-I (DCARDs: 1–238): 50

ppp8L: ADP-Mg2+, RIG-I (DCARDs: 1–238) at 2.5 mg ml�1 was preassem- bled with 50 ppp8L at 50 mM and with 2.5 mM ADP, 2.5 mM MgCl2, 2.5 mM BeCl2, 12.5 mM NaF on ice for 1 hr. The complex solution was

then mixed with equal volumes of precipitating solution (0.1 M Bicine [pH

9.0], 26%–28% polyethylene glycol 6,000) and then grown at 13�C. Crystals also grew into needle clusters within 3 days and were harvested within

2 weeks. Crystals were soaked in a cryoprotecting solution containing

0.1 M Bicine (pH 9.0), 30% polyethylene glycol 6,000 briefly before being

flash frozen with liquid nitrogen. Diffraction intensities were recorded at

NE-CAT beamline ID-24 at the Advanced Photon Source (Argonne National

Laboratory, Argonne, IL). Integration, scaling, and merging of the intensities

were carried out by using the programs XDS (Kabsch, 2010) and SCALA

(Evans, 2006).

Structure Determination and Refinement

The structures were determined through molecular replacement with the

program Phaser (McCoy, 2007) by using the structure of RIG-I (DCARDs: 1–

229): 50 OH-GC10 (PDB: 2ykg) as search model. Refinement cycles were carried out by using Phenix Refine (Adams et al., 2010) and REFMAC5 (Mur-

shudov et al., 1997) with the TLS (translation, liberation, screw-rotation

displacement) refinement option with four TLS groups (HEL1: aa 239–455,

HEL2-HEL2i: aa 456–795, CTD: aa 796–922, and dsRNA). Refinement cycles

were interspersed with model rebuilding by using Coot (Emsley and Cowtan,

1986 Structure 20, 1983–1988, November 7, 2012 ª2012 Elsevier Ltd All rights reserved

2004). The quality of the structures was analyzed

by using MolProbity (Davis et al., 2007). A

summary of the data collection and structure

refinement statistics is given in Table 1. Figures

were prepared by using the program Pymol (De-

Lano, 2002).

Sedimentation Velocity Studies

Samples were prepared by mixing 3 mM 50 Dy- light 547-U10:A10 duplex RNA with 7.5 mM of

full-length RIG-I protein in a buffer containing

25 mM HEPES, 150 mM NaCl, 0.5% glycerol,

5 mM b-ME, 2.5 mM MgCl2 (pH 7.4), in addition

to the respective ATP analogs (ADP-AlFx:

2.5 mM ADP, 2.5 mM MgCl2, 2.5 mM AlCl3,

12.5 mM NaF; ADP-BeF3: 2.5 mM ADP,

2.5 mM MgCl2, 2.5 mM BeCl3, 12.5 mM NaF;

ADP: 2.5 mM ADP, 2.5 mM MgCl2). The samples were then incubated on

ice for 1 hr. SV experiments were performed at 20�C in a Beckman Optima XL-I analytical ultracentrifuge. A four position AN 60 Ti rotor, together

with Epon 12 mm double-sector centerpieces, was used at 40,000 rpm.

Radial absorption scans were measured at 547 nm with a radial increment

of 0.003 cm. Data analyses were performed in Sedfit 8.0 (http://www.

analyticalultracentrifugation.com) (Schuck et al., 2002). Sedimentation

coefficients at the experimental temperature, buffer density, and viscosity

were corrected to standard conditions (s20, w) using the program SEDNTERP

(http://jphilo.mailway.com).

ACCESSION NUMBERS

The atomic coordinates and structure factors of the ternary complex of RIG-I

(DCARDs: 1–238): 50 ppp8L: ADP-Mg2+ have been deposited with the RCSB Protein Data Bank under the accession code 4ay2.

SUPPLEMENTAL INFORMATION

Supplemental Information includes four figures, one table, and one movie

and can be found with this article online at http://dx.doi.org/10.1016/

j.str.2012.08.029.

ACKNOWLEDGMENTS

We thank members of the A.M.P. Lab for their generous help and insightful

discussions. We thank Dr. Steve Ding for providing 50 Dylight 547-U10 RNA. We thank scientists from APS NECAT 24-ID for the beamline access and

technical support. This research was funded by the Howard Hughes Medical

Institute and NIH Grant R01AI089826. D.L. is a postdoctoral associate and

A.M.P. is an investigator with the Howard Hughes Medical Institute.

Figure 3. Sequential Activation of RIG-I by RNA and ATP

(A) Schematic representation of RIG-I protein.

(B) ADP-AlFx binding induced conformational changes of RIG-I. Conforma-

tional changes upon ADP-AlFx binding is modeled based on the following

crystal structures: human RIG-I:dsRNA binary complex (PDB: 2ykg), duck

RIG-I apo enzyme (PDB: 4a2w), and duck RIG-I:dsRNA:ADP-AlFx ternary

complex (PDB: 4a36) (Kowalinski et al., 2011; Luo et al., 2011). The binding of

ADP-AlFx (blue) causes the helicase domain to close and moves the CTD

(red) and HEL2i (green) toward each other. This directional movement prob-

ably allows the CARDs (orange) to be released from HEL2i which otherwise

would clash with CTD (circled box). As a result, the structure is likely to

reorganize, reorienting the relative positions of the CARDs and HEL2i. This

structural arrangement may allow the CARDs to gain access to poly-

ubiquitins, making it available for MAVS activation (Jiang et al., 2012; Zeng

et al., 2010).

See also Movie S1.

Structure

Structure of RIG-I, 50 ppp-dsRNA, and ADP

Received: August 17, 2012

Revised: August 17, 2012

Accepted: August 22, 2012

Published online: September 27, 2012

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