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• W2135087277 abstract "The RIG-I-like receptors (RLRs), RIG-I and MDA5, recognize single-stranded RNA with 5′ triphosphates and double-stranded RNA (dsRNA) to initiate innate antiviral immune responses. LGP2, a homolog of RIG-I and MDA5 that lacks signaling capability, regulates the signaling of the RLRs. To establish the structural basis of dsRNA recognition by the RLRs, we have determined the 2.0-Å resolution crystal structure of human LGP2 C-terminal domain bound to an 8-bp dsRNA. Two LGP2 C-terminal domain molecules bind to the termini of dsRNA with minimal contacts between the protein molecules. Gel filtration chromatography and analytical ultracentrifugation demonstrated that LGP2 binds blunt-ended dsRNA of different lengths, forming complexes with 2:1 stoichiometry. dsRNA with protruding termini bind LGP2 and RIG-I weakly and do not stimulate the activation of RIG-I efficiently in cells. Surprisingly, full-length LGP2 containing mutations that abolish dsRNA binding retained the ability to inhibit RIG-I signaling. The RIG-I-like receptors (RLRs), RIG-I and MDA5, recognize single-stranded RNA with 5′ triphosphates and double-stranded RNA (dsRNA) to initiate innate antiviral immune responses. LGP2, a homolog of RIG-I and MDA5 that lacks signaling capability, regulates the signaling of the RLRs. To establish the structural basis of dsRNA recognition by the RLRs, we have determined the 2.0-Å resolution crystal structure of human LGP2 C-terminal domain bound to an 8-bp dsRNA. Two LGP2 C-terminal domain molecules bind to the termini of dsRNA with minimal contacts between the protein molecules. Gel filtration chromatography and analytical ultracentrifugation demonstrated that LGP2 binds blunt-ended dsRNA of different lengths, forming complexes with 2:1 stoichiometry. dsRNA with protruding termini bind LGP2 and RIG-I weakly and do not stimulate the activation of RIG-I efficiently in cells. Surprisingly, full-length LGP2 containing mutations that abolish dsRNA binding retained the ability to inhibit RIG-I signaling. The innate immune response is the first line of defense against invading pathogens; it is the ubiquitous system of defense against microbial infections (1Janeway Jr., C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Crossref PubMed Scopus (6061) Google Scholar). Toll-like receptors (TLRs) 3The abbreviations used are: TLR, Toll-like receptor; CTD, C-terminal domain; 5′ ppp ssRNA, 5′-triphosphorylated single-stranded RNA; dsRNA, double-stranded RNA; RLR, RIG-I-like receptor; IFN, interferon; CARD, caspase recruiting domain; bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; siRNA, small interfering RNA. 3The abbreviations used are: TLR, Toll-like receptor; CTD, C-terminal domain; 5′ ppp ssRNA, 5′-triphosphorylated single-stranded RNA; dsRNA, double-stranded RNA; RLR, RIG-I-like receptor; IFN, interferon; CARD, caspase recruiting domain; bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; siRNA, small interfering RNA. and RIG-I (retinoic acid-inducible gene 1)-like receptors (RLRs) play key roles in innate immune response toward viral infection (2Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8554) Google Scholar, 3Yoneyama M. Fujita T. J. Biol. Chem. 2007; 282: 15315-15318Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 4Pichlmair A. Reis e Sousa C. Immunity. 2007; 27: 370-383Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar, 5Thompson A.J. Locarnini S.A. Immunol. Cell Biol. 2007; 85: 435-445Crossref PubMed Scopus (202) Google Scholar). Toll-like receptors TLR3, TLR7, and TLR8 sense viral RNA released in the endosome following phagocytosis of the pathogens (6Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6586) Google Scholar). RIG-I-like receptors RIG-I and MDA5 detect viral RNA from replicating viruses in infected cells (3Yoneyama M. Fujita T. J. Biol. Chem. 2007; 282: 15315-15318Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 7Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3068) Google Scholar, 8Takeuchi O. Akira S. Curr. Opin. Immunol. 2008; 20: 17-22Crossref PubMed Scopus (450) Google Scholar). Stimulation of these receptors leads to the induction of type I interferons (IFNs) and other proinflammatory cytokines, conferring antiviral activity to the host cells and activating the acquired immune responses (4Pichlmair A. Reis e Sousa C. Immunity. 2007; 27: 370-383Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar, 9Akira S. Takeda K. Kaisho T. Nat. Immunol. 2001; 2: 675-680Crossref PubMed Scopus (3898) Google Scholar).RIG-I discriminates between viral and host RNA through specific recognition of the uncapped 5′-triphosphate of single-stranded RNA (5′ ppp ssRNA) generated by viral RNA polymerases (10Hornung V. Ellegast J. Kim S. Brzozka K. Jung A. Kato H. Poeck H. Akira S. Conzelmann K.K. Schlee M. Endres S. Hartmann G. Science. 2006; 314: 994-997Crossref PubMed Scopus (1852) Google Scholar, 11Pichlmair A. Schulz O. Tan C.P. Naslund T.I. Liljestrom P. Weber F. Reis e Sousa C. Science. 2006; 314: 997-1001Crossref PubMed Scopus (1739) Google Scholar). In addition, RIG-I also recognizes double-stranded RNA generated during RNA virus replication (7Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3068) Google Scholar, 12Kato H. Takeuchi O. Sato S. Yoneyama M. Yamamoto M. Matsui K. Uematsu S. Jung A. Kawai T. Ishii K.J. Yamaguchi O. Otsu K. Tsujimura T. Koh C.S. Reis e Sousa C. Matsuura Y. Fujita T. Akira S. Nature. 2006; 441: 101-105Crossref PubMed Scopus (2845) Google Scholar). Transfection of cells with synthetic double-stranded RNA stimulates the activation of RIG-I (13Marques J.T. Devosse T. Wang D. Zamanian-Daryoush M. Serbinowski P. Hartmann R. Fujita T. Behlke M.A. Williams B.R. Nat. Biotechnol. 2006; 24: 559-565Crossref PubMed Scopus (321) Google Scholar, 14Takahasi K. Yoneyama M. Nishihori T. Hirai R. Kumeta H. Narita R. Gale Jr., M. Inagaki F. Fujita T. Mol. Cell. 2008; 29: 428-440Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Synthetic dsRNA mimics, such as polyinosinic-polycytidylic acid (poly(I·C)), can activate MDA5 when introduced into the cytoplasm of cells. Digestion of poly(I·C) with RNase III transforms poly(I·C) from a ligand for MDA5 into a ligand for RIG-I, suggesting that MDA5 recognizes long dsRNA, whereas RIG-I recognizes short dsRNA (15Kato H. Takeuchi O. Mikamo-Satoh E. Hirai R. Kawai T. Matsushita K. Hiiragi A. Dermody T.S. Fujita T. Akira S. J. Exp. Med. 2008; 205: 1601-1610Crossref PubMed Scopus (1132) Google Scholar). Studies of RIG-I and MDA5 knock-out mice confirmed the essential roles of these receptors in antiviral immune responses and demonstrated that they sense different sets of RNA viruses (12Kato H. Takeuchi O. Sato S. Yoneyama M. Yamamoto M. Matsui K. Uematsu S. Jung A. Kawai T. Ishii K.J. Yamaguchi O. Otsu K. Tsujimura T. Koh C.S. Reis e Sousa C. Matsuura Y. Fujita T. Akira S. Nature. 2006; 441: 101-105Crossref PubMed Scopus (2845) Google Scholar, 16Gitlin L. Barchet W. Gilfillan S. Cella M. Beutler B. Flavell R.A. Diamond M.S. Colonna M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8459-8464Crossref PubMed Scopus (898) Google Scholar).RIG-I and MDA5 contain two caspase recruiting domains (CARDs) at their N termini, a DEX(D/H) box RNA helicase domain, and a C-terminal regulatory or repressor domain (CTD). The helicase domain and the CTD are responsible for viral RNA binding, whereas the CARDs are required for signaling (3Yoneyama M. Fujita T. J. Biol. Chem. 2007; 282: 15315-15318Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 8Takeuchi O. Akira S. Curr. Opin. Immunol. 2008; 20: 17-22Crossref PubMed Scopus (450) Google Scholar). The current model of RIG-I activation suggests that under resting conditions RIG-I is in a suppressed conformation, and viral RNA binding triggers a conformation change that leads to the exposure of the CARDs for the recruitment of the downstream protein IPS-1 (also known as MAVS, Cardif, or VISA) (14Takahasi K. Yoneyama M. Nishihori T. Hirai R. Kumeta H. Narita R. Gale Jr., M. Inagaki F. Fujita T. Mol. Cell. 2008; 29: 428-440Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 17Yoneyama M. Fujita T. Immunity. 2008; 29: 178-181Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Limited proteolysis of the RIG-I·dsRNA complex showed that RIG-I residues 792-925 of the CTD are involved in dsRNA and 5′ ppp ssRNA binding (14Takahasi K. Yoneyama M. Nishihori T. Hirai R. Kumeta H. Narita R. Gale Jr., M. Inagaki F. Fujita T. Mol. Cell. 2008; 29: 428-440Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). The CTD of RIG-I overlaps with the C terminus of the previously identified repressor domain (18Saito T. Hirai R. Loo Y.M. Owen D. Johnson C.L. Sinha S.C. Akira S. Fujita T. Gale Jr., M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 582-587Crossref PubMed Scopus (570) Google Scholar). The structures of RIG-I and LGP2 (laboratory of genetics and physiology 2) CTD in isolation have been determined by x-ray crystallography and NMR spectroscopy (14Takahasi K. Yoneyama M. Nishihori T. Hirai R. Kumeta H. Narita R. Gale Jr., M. Inagaki F. Fujita T. Mol. Cell. 2008; 29: 428-440Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 19Cui S. Eisenacher K. Kirchhofer A. Brzozka K. Lammens A. Lammens K. Fujita T. Conzelmann K.K. Krug A. Hopfner K.P. Mol. Cell. 2008; 29: 169-179Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 20Pippig D.A. Hellmuth J.C. Cui S. Kirchhofer A. Lammens K. Lammens A. Schmidt A. Rothenfusser S. Hopfner K.P. Nucleic Acids Res. 2009; 10.1093/nar/gkp059PubMed Google Scholar). A large, positively charged surface on RIG-I recognizes the 5′ triphosphate group of viral ssRNA (14Takahasi K. Yoneyama M. Nishihori T. Hirai R. Kumeta H. Narita R. Gale Jr., M. Inagaki F. Fujita T. Mol. Cell. 2008; 29: 428-440Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 19Cui S. Eisenacher K. Kirchhofer A. Brzozka K. Lammens A. Lammens K. Fujita T. Conzelmann K.K. Krug A. Hopfner K.P. Mol. Cell. 2008; 29: 169-179Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar). RNA binding studies by titrating RIG-I CTD with dsRNA and 5′ ppp ssRNA suggested that overlapping sets of residues on this charged surface are involved in RNA binding (14Takahasi K. Yoneyama M. Nishihori T. Hirai R. Kumeta H. Narita R. Gale Jr., M. Inagaki F. Fujita T. Mol. Cell. 2008; 29: 428-440Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Mutagenesis of several positively charged residues on this surface either reduces or disrupts RNA binding by RIG-I, and these mutations also affect the induction of IFN-β in vivo (14Takahasi K. Yoneyama M. Nishihori T. Hirai R. Kumeta H. Narita R. Gale Jr., M. Inagaki F. Fujita T. Mol. Cell. 2008; 29: 428-440Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 19Cui S. Eisenacher K. Kirchhofer A. Brzozka K. Lammens A. Lammens K. Fujita T. Conzelmann K.K. Krug A. Hopfner K.P. Mol. Cell. 2008; 29: 169-179Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar). However, the exact nature of how the RLRs recognize viral RNA and how RNA binding activates these receptors remains to be established.LGP2 is a homolog of RIG-I and MDA5 that lacks the CARDs and thus has no signaling capability (21Rothenfusser S. Goutagny N. DiPerna G. Gong M. Monks B.G. Schoenemeyer A. Yamamoto M. Akira S. Fitzgerald K.A. J. Immunol. 2005; 175: 5260-5268Crossref PubMed Scopus (481) Google Scholar, 22Yoneyama M. Kikuchi M. Matsumoto K. Imaizumi T. Miyagishi M. Taira K. Foy E. Loo Y.M. Gale Jr., M. Akira S. Yonehara S. Kato A. Fujita T. J. Immunol. 2005; 175: 2851-2858Crossref PubMed Scopus (1262) Google Scholar). The expression of LGP2 is inducible by dsRNA or IFN treatment as well as virus infection (21Rothenfusser S. Goutagny N. DiPerna G. Gong M. Monks B.G. Schoenemeyer A. Yamamoto M. Akira S. Fitzgerald K.A. J. Immunol. 2005; 175: 5260-5268Crossref PubMed Scopus (481) Google Scholar). Overexpression of LGP2 inhibits Sendai virus and Newcastle disease virus signaling (21Rothenfusser S. Goutagny N. DiPerna G. Gong M. Monks B.G. Schoenemeyer A. Yamamoto M. Akira S. Fitzgerald K.A. J. Immunol. 2005; 175: 5260-5268Crossref PubMed Scopus (481) Google Scholar). When coexpressed with RIG-I, LGP2 can inhibit RIG-I signaling through the interaction of its CTD with the CARD and the helicase domain of RIG-I (18Saito T. Hirai R. Loo Y.M. Owen D. Johnson C.L. Sinha S.C. Akira S. Fujita T. Gale Jr., M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 582-587Crossref PubMed Scopus (570) Google Scholar). LGP2 could suppress RIG-I signaling by three possible ways (23Komuro A. Bamming D. Horvath C.M. Cytokine. 2008; 43: 350-358Crossref PubMed Scopus (106) Google Scholar): 1) binding RNA with high affinity, thereby sequestering RNA ligands from RIG-I; 2) interacting directly with RIG-I to block the assembly of the signaling complex; and 3) competing with IKKi (IκB kinase ϵ) in the NF-κB signaling pathway for a common binding site on IPS-1. To elucidate the structural basis of dsRNA recognition by the RLRs, we have crystallized human LGP2 CTD (residues 541-678) bound to an 8-bp double-stranded RNA and determined the structure of the complex at 2.0 Å resolution. The structure revealed that LGP2 CTD binds to the termini of dsRNA. Mutagenesis and functional studies showed that dsRNA binding is likely not required for the inhibition of RIG-I signaling by LGP2.EXPERIMENTAL PROCEDURESProtein Expression and Purification—DNAs encoding the C-terminal domains of human LGP2 (residues 541-678), RIG-I (residues 802-925), and MDA5 (residues 892-1017) were cloned into expression vector pET22b(+) (Novagen). All of the cloned DNA sequences were confirmed by plasmid DNA sequencing. The proteins were expressed in Escherichia coli strain BL21(DE3) by induction at A600 = 0.6-0.8 with 0.5 mm isopropyl-β-d-thiogalactoside overnight at 15 °C. The cells were lysed by sonication, and the proteins were purified by batch method using His-Select nickel affinity resin (Sigma-Aldrich) in a buffer containing 20 mm Tris, 150 mm NaCl, at pH 7.5 (buffer A). After incubation for 2 h, the nickel beads were collected and washed three times with 10 volumes of buffer A containing 10 mm imidazole, and the proteins were eluted with buffer A containing 250 mm imidazole. The proteins were further purified by gel filtration chromatography on a Superdex75 (1.6 × 60) column (GE Healthcare) eluted with buffer A. To form the complex with dsRNA, LGP2 CTD were mixed with an 8-bp dsRNA at a molar ratio of 1:1, and the 2:1 LGP2·dsRNA complex was purified by gel filtration chromatography on a Superdex75 (1.6 × 60) column. Mutants of full-length LGP2 and LGP2 CTD were generated using a QuikChange mutagenesis kit (Stratagene). The sequences of the mutants were confirmed by plasmid DNA sequencing. The mutant proteins were expressed and purified the same way as the native protein.Crystallization, Data Collection, and Structure Determination—Purified LGP2·dsRNA complex was concentrated to ∼30 mg ml-1 in a buffer containing 20 mm Tris, 150 mm NaCl, and 4 mm dithiothreitol at pH 7.5. The complex was crystallized with 16-18% (w/v) PEG3350 in a buffer containing 0.2 m (NH4)2SO4, 0.1 m Tris-HCl at pH 8.5. The crystals were transferred stepwise from the mother liquor to a cryoprotectant containing 25% (v/v) glycerol and flash frozen in liquid nitrogen. The LGP2·dsRNA complex crystallized in space group C2, with cell dimensions: a = 116.46 Å, b = 54.19 Å, c = 67.20 Å, and β = 97.26°. The crystallographic asymmetric unit contains one 2:1 LGP2·dsRNA complex. Diffraction data were collected using a Rigaku RAXIS IV2+ image plate detector mounted on a Rigaku Micromax-007HF generator and processed with the HKL package (24Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38368) Google Scholar). Statistics of data collection and refinement are shown in Table 1.TABLE 1Data collection and refinement statisticsLGP2/dsRNAData collectionSpace groupC2Cell dimensionsa, b, c (Å)116.46, 54.19, 67.20α, β, γ (°)90.00, 97.26, 90.00Resolution (Å)50-2.00 (2.07-2.00)aThe values in parentheses are for highest resolution shell.Rmerge4.6 (33.6)I/σI46.2 (5.1)Completeness (%)99.6 (98.6)Redundancy3.6 (3.5)RefinementResolution (Å)50-2.00No. reflections27234Rwork/Rfree21.3/25.4No. atomsProtein2167RNA340Zinc ion2Water215B factorsProtein46.6RNA43.5Zinc ion51.5Water52.2Root mean square deviationsBond lengths (Å)0.008Bond angles (°)1.43a The values in parentheses are for highest resolution shell. Open table in a new tab The crystal structure of the LGP2·dsRNA complex was determined by molecular replacement with MOLREP in the CCP4 suite (25Collaborative Computational ProjectActa Crystallogr. D Biol. Crystallogr. 1994; 50 (number 4): 760-763Crossref PubMed Scopus (19707) Google Scholar) using the crystal structure of RIG-I CTD as search model (Protein Data Bank code 2QFB, chain A). The model was rebuilt using O (26Jones T.A. Kjeldgaard M. Methods Enzymol. 1997; 277: 173-208Crossref PubMed Scopus (504) Google Scholar). After several rounds of rebuilding and refinement with CNS (27Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16930) Google Scholar), the electron density for the dsRNA became apparent. An 8-bp dsRNA from the TLR3·dsRNA complex structure (Protein Data Bank code 3CIY) was docked into the electron density map and rebuilt with O. The complex structure was refined by several rounds of positional, simulated annealing, and individual B-factor refinement using CNS followed by manual remodeling after each round of refinement.RNA Binding Studies by Gel Filtration Chromatography—RNAs used in the binding studies were chemically synthesized by IDT (Coralville, IA) or by in vitro transcription using T7 RNA polymerase. The sequences of the RNAs are shown in supplemental Table S2. Double-stranded RNAs were generated by heating the ssRNA at 95 °C for 5 min and annealing at room temperature for 30 min. Each dsRNA was mixed with excess protein (RNA to protein molar ratio of 1:3), and 100 μl of samples were injected over a Superdex200 (10/300 GL) column (GE Healthcare) eluted with buffer A. The column was calibrated with a set of protein standards for gel filtration chromatography (Bio-Rad) to ensure accurate estimation of the molecular weight of the LGP2·dsRNA complexes.RNA Binding Studies by Fluorescence Anisotropy—Cy3-labeled RNA probes were heated to anneal with their complementary RNA to form dsRNA. Fluorescence measurements were performed at room temperature using a PerkinElmer Life Sciences luminescence spectrometer LS55. The excitation and emission wavelengths are 540 and 565 nm, respectively, with an integration time of 2 s and slit width of 5 nm. Purified LGP2, RIG-I, and MDA5 CTD were titrated into the dsRNA in buffer A and mixed by magnetic stirring. Total volume of protein added was less than 3% of the final sample volume. The represented anisotropy values used to calculate the affinity are the average of 10 measurements. The binding data were analyzed by nonlinear least square fitting using KaleidaGraph software (Synergy Software, Reading, PA). The Hill equation, ΔA = Bmax Xn/(Xn + Kdn) was used to determine the dissociation constant (Kd). In this equation, ΔA is the anisotropy change caused by the ligand binding, Bmax is the maximum anisotropy change, X is the total concentration of the input protein, and the exponential term n is the Hill coefficient. Binding studies for the mutants of LGP2 CTD were carried out under similar conditions.Analytical Ultracentrifugation—To verify the stoichiometry of LGP2 binding to dsRNA, LGP2 CTD complexes with the 8-bp dsRNA, the 24-bp dsRNA, and the hairpin RNA were analyzed by sedimentation velocity. 400-μl samples in 20 mm Tris-HCl buffer at pH 7.5, 150 mm NaCl, 10 mm β-mercaptoethanol were spun overnight at 48,000 rpm at 20 °C in a Beckman XL-I using absorbance optics at 302 nm. The data were analyzed by the program Sedfit using the c(s) and c(M) models to determine differential sedimentation coefficient and apparent mass distributions, respectively. The 8-bp dsRNA·LGP2 complex gave an estimated molecular mass value similar to that of a 2:1 complex, whereas the hairpin RNA·LGP2 complex had an estimated molecular mass consistent with a 1:1 complex. The 24-bp dsRNA·LGP2 complex sedimented as a single peak, but the estimated mass could indicate either a 2:1 or a 2:2 complex. To resolve the stoichiometry, a sedimentation equilibrium experiment was performed on the 24-bp dsRNA·LGP2 complex. 100-μl samples of purified 24-bp dsRNA·LGP2 complex at concentrations of 10, 33.3, and 100 μm were spun at 17,000, 21,000, and 30,000 rpm at 20 °C until equilibrium was reached, and scanned at 297 and 305 nm. The data were trimmed using Win-Reedit and globally analyzed using WinNonlin. The global analysis of nine data sets revealed that the data could be described by a single species with a reduced buoyant molecular weight of 1.836 (at a speed of 17,000 rpm). To convert to experimental molecular weight, the partial specific volume must be known; for a protein·RNA complex this value corresponds to the weight average of each component in the sedimenting species. The partial specific volume for LGP2 CTD alone was calculated to be 0.7328 based on sequence using Sednterp, and that for the 24-bp dsRNA was calculated to be 0.5688 using the NucProt server. Both 2:1 and 2:2 complexes were considered, with weight average partial specific volumes of 0.6813 or 0.6566, respectively. The data were consistent with a 2:1 complex but were not consistent with either a 1:1 or a 2:2 complex.Reporter Gene Assays—Actively growing HEK 293T cells were plated in CoStar White 96-well plates at 4.4 × 104 cells ml-1 for transfection. When the cells were ∼60-80% confluent, they were transfected with a mixture of Lipofectamine 2000 reagent (Invitrogen) and constant amounts of the reporter plasmids IFN-β Luc (30 ng) or pNiFty Luc (15 ng; Invivogen), which contain the firefly luciferase gene, phRL-TK (5 ng, Promega), and phRIG-I (0.5 ng; Invivogen). In the functional assays of LGP2, phLGP2 (1.0 ng; Invivogen) were cotransfected with the same amount of phRIG-I and the reporter plasmids as described above. The cells were incubated for 24 h to allow expression from the plasmids. The dsRNA ligands were then transfected into the cells at a final concentration of 0.5 or 0.2 μm as indicated in the figure legend. After 12 h of incubation, the cells were analyzed using the Dual Glo luciferase assay system reagents (Promega), quantifying luminescence with the FLU-Ostar OPTIMA Plate Reader (BMG Labtech.). The ratios of firefly luciferase over Renilla luciferase were plotted. The RNA ligands used were purified by preparative denaturing polyacrylamide gel electrophoresis or gel filtration chromatography.Western Blot—293T cells transfected with wild type LGP2 or its mutants were lysed with the passive lysis buffer (Promega) and sonicated to degrade chromosomal DNA. Equal amounts of proteins from each sample, as determined by staining with Coomassie Blue, were separated on NuPAGE 4-12% (w/v) bistris gels (Invitrogen) and blotted onto polyvinylidene difluoride membrane. Affinity-purified rabbit antibody against human LGP2 (Proteintec) was used as primary antibody. The blots were developed with peroxidase-conjugated secondary antibodies and an ECL Plus Western blotting detection system (GE Healthcare).RESULTSOverall Structure of the LGP2·dsRNA Complex—The structure of LGP2 CTD in complex with dsRNA was determined by molecular replacement using the crystal structure of RIG-I CTD (19Cui S. Eisenacher K. Kirchhofer A. Brzozka K. Lammens A. Lammens K. Fujita T. Conzelmann K.K. Krug A. Hopfner K.P. Mol. Cell. 2008; 29: 169-179Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar) as a search model. The crystallographic asymmetric unit contains a 2:1 complex between LGP2 CTD and the 8-bp dsRNA (Fig. 1A). The refined model includes residues 546-672 of LGP2 in molecule A, residues 544-678 of LGP2 plus a five-residue His tag in molecule B, and the 8-bp dsRNA. The complex exhibits pseudo 2-fold symmetry. The root mean square deviation between the two LGP2 CTD molecules is 0.45 Å. The following discussion is based on the structure of the complex between LGP2 (A) and the dsRNA caused by better defined electron density of the molecule. The structure of LGP2 CTD in the complex is shown in Fig. 1B.The 8-bp dsRNA binds to a deep groove between two LGP2 CTD molecules, making primary contacts with LGP2 through the two blunt ends (Fig. 1A). Apart from two hydrogen bonds that involve residue Lys650, the two protein molecules do not interact with each other, suggesting that formation of the protein dimer is not required for RNA binding. The dsRNA adopts a typical A-form structure with slight distortions at the two ends. The binding surfaces between LGP2 and dsRNA show a high degree of shape and charge complementarity (Fig. 1, C and D). LGP2 interacts with the phosphate backbone of the RNA through extensive electrostatic interactions and hydrogen bonding. The exposed terminal GC base pair interacts with LGP2 through extensive hydrophobic interactions. The total buried surface area at the LGP2 dsRNA interface is ∼1540 Å2, with major contributions from the first six nucleotides at the 5′ end of one RNA strand (buried surface area, ∼1020 Å2) and minor contributions from the two nucleotides from the 3′ end of the complementary strand (buried surface area, ∼520 Å2). The calculated shape correlation statistics (Sc, a measure of the degree that two contacting surfaces are geometrically matched) is 0.70, where an Sc value of 1.0 indicates a perfect fit (28Lawrence M.C. Colman P.M. J. Mol. Biol. 1993; 234: 946-950Crossref PubMed Scopus (1097) Google Scholar). The buried surface area and shape complementarity between LGP2 and dsRNA are comparable with typical antibody·peptide antigen complexes, which have an average buried surface area of 1430 Å2 and an Sc of 0.75 (29Li P. Huey-Tubman K.E. Gao T. Li X. West Jr., A.P. Bennett M.J. Bjorkman P.J. Nat. Struct. Mol. Biol. 2007; 14: 381-387Crossref PubMed Scopus (56) Google Scholar).Structure of LGP2 C-terminal Domain—Although the amino acid sequences of LGP2 and RIG-I CTD are only 25% identical (supplemental Fig. S1), the structures of LGP2 and RIG-I CTD are highly conserved (Fig. 1E). The root mean square deviation between the 102 conserved Cα atoms in the two proteins is only 0.90 Å. LGP2 CTD contains a three-stranded (β1, β2, and β9) antiparallel β-sheet near its N terminus and a four-stranded (β5, β6, β7, and β8) antiparallel β-sheet in the middle (Fig. 1B). The two β-sheets are connected by a β-hairpin formed by strands β3 and β4 and two short α-helices. Four conserved cysteine residues (Cys556, Cys559, Cys612, and Cys615) in the two loops connecting strands β1-β2 and β6-β7 make additional connections between the two β-sheets by coordinating a zinc ion (Fig. 1B). The eight residues near the C terminus of LGP2 form a well defined α-helix, whereas the corresponding helix in RIG-I is only four residues long. The RNA-binding site of LGP2 is located at the large concave surface defined by the β-sheet containing strands β5 to β8, the β-hairpin, and the three loops connecting β5 to β6, β8 to β9, and β9 to the C-terminal helix (Fig. 1, B and C).The major difference between the crystal structure of LGP2 and RIG-I CTD occurs in the long loop (loop5-6) connecting strands β5 and β6 (Fig. 1E). NMR structure of RIG-I CTD showed that this loop is flexible (14Takahasi K. Yoneyama M. Nishihori T. Hirai R. Kumeta H. Narita R. Gale Jr., M. Inagaki F. Fujita T. Mol. Cell. 2008; 29: 428-440Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). The structure of LGP2 CTD in isolation showed that this loop is not ordered (20Pippig D.A. Hellmuth J.C. Cui S. Kirchhofer A. Lammens K. Lammens A. Schmidt A. Rothenfusser S. Hopfner K.P. Nucleic Acids Res. 2009; 10.1093/nar/gkp059PubMed Google Scholar). The ordered structure of this loop observed in the LGP2·dsRNA complex structure (Fig. 1E) is most likely due to the binding of dsRNA, especially the hydrophobic interactions with the two bases at the blunt end of the dsRNA.Structural Basis of dsRNA Recognition by LGP2—Unlike TLR3 that binds primarily to the phosphate backbone of long dsRNA (30Liu L. Botos I. Wang Y. Leonard J.N. Shiloach J. Segal D.M. Davies D.R. Science. 2008; 320: 379-381Crossref PubMed Scopus (560) Google Scholar), LGP2 binds specifically to the ends of dsRNA (Fig. 2A and Table 2). The exposed terminal GC base pairs are recognized through hydrophobic interactions that involve residues Val632, Leu621, Val595, Ile597, Phe601, and Trp604 (Fig. 2, B and C, and Table 2). However, there are significant differences in the interactions between LGP2 CTD and the two RNA strands at each of the dsRNA terminus. The two hydroxyl groups of the ribose at the 3′ end of the two RNA strands interact with LGP2 through a network of five direct hydrogen bonds with the side chains of Glu573, His576, and Trp604 (Fig. 2B), as well as a solvent-mediated hydrogen bond with the backbone amine gro" @default.
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• W2135087277 title "The RIG-I-like Receptor LGP2 Recognizes the Termini of Double-stranded RNA" @default.
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