doi:10.1038/nature10537 a Binding fraction 1.2 1.0 0.8 0.6 0.4 0.2 b 0.0 1 10 100 1000 [Protein] (nm) Supplementary Figure 1. Stoichiometry of RIG-I helicase-rd bound to dsrna. (a) 14 base pair dsrna binding by RIG-I helicase-rd (green squares), RD (red triangles), and helicase (black circles) using fluorescence anisotropy titrations. The fit to a quadratic equation (1:1 stoichiometry) yielded dissociation constant (K d ) values: 436 ± 121 nm for the helicase domain, 2.6 ± 1.2 nm for the RD, and <0.1 nm for helicase-rd. Binding of the helicase-rd at low RNA concentrations (1 and 2 nm), revealed a K d = 0.05 ± 0.02 nm. (b) Chromatogram of size exclusion chromatography showing RIG-I helicase-rd (blue), 14 base pair dsrna (green) and helicase-rd in complex to dsrna (red). Helicase-RD dsrna complex elutes at a larger volume (14.3ml) compared to the free helicase-rd (13.7ml). WWW.NATURE.COM/NATURE 1
Supplementary Figure 2. Confirmation of RNA sequence using 5-Iodo-Uridine substituted RNA. The sequence and orientation of the dsrna in the structure was confirmed using data collected from a crystal substituted with 5-Iodo-uridine at position 12 of the dsrna. Crystals of RIG-I helicase-rd, ADP BeF 3 and 14 base pair dsrna substituted with 5-Iodo-uridine at position 12 were grown under identical conditions as the native complex. (2F observed -F calculated ) electron density maps at 3.1 Å resolution calculated using the protein alone contoured at 5σ is shown in green. The peak corresponds to an iodine atom at the 5 position of a uridine base. While the last two base pairs of the RNA are disordered, the second 5-Iodo-uridine could be located at the U12 position of the opposite strand but at a lower electron density (data not shown). 2 WWW.NATURE.COM/NATURE
240 250 260 270 280 290 300 RIG-I PFKP R N YQ LELALPAMKGKNTIICAP TG C GKT FVSLLICEHHLKKFP..Q.GQKGKVVFFANQIPVYE Q Q MDA5 ELQL R P YQ MEVAQPALEGKNIIICLP TG S GKT RVAVYIAKDHLDKKK..KASEPGKVIVLVNKVLLVE Q L LGP2.MEL R S YQ WEVIMPALEGKNIIIWLP TG A GKT RAAAYVAKRHLET...VDGAKVVVLVNRVHLVT Q H Dicer IYTP R K YQ VELLEAALDHN.TIVCLN TG S GKT FIAVLLTKELSYQIRGDFSRNGKRTVFLVNSANQVA Q Q FANCM NCPV R D YQ LHISRAALFCN.TLVCLP TG L GKT FIAAVVMYNFYRWFP..S...GKVVFMAPTKPLVT Q Q Hef...P R I YQ EVIYAKCKETN.CLIVLP TG L GKT LIAMMIAEYRLTKYG...GKVLMLAPTKPLVL Q H Q I Ia. 310 320 330 340 350 360 RIG-I K.SVFSKYFERHGYRVTGISGATAENVP.VEQIVENNDIIIL T PQILVNN L KKGT...IPSLSIFTLM MDA5 FRKEFQPFLKKW.YRVIGLSGDTQLKIS.FPEVVKSCDIIIS T AQILENS L LNLENGEDAGVQLSDFSLI LGP2 G.EEFRRMLDGR.WTVTTLSGDMGPRAG.FGHLARCHDLLIC Ib T AELLQMA L TSPE..EEEHVELTVFSLI Dicer V.SAVRTHSDLKVGEYSNLEVNASWTKERWNQEFTKHQVLIM T CYVALNV L KNGY...LSLSDINLL FANCM I.EACYQVMGIPQSHMAEMTGSTQAST..RKEIWCSKRVLFL T PQVMVND L SRGA...CPAAEIKCL Hef A.ESFRRLFNLPPEKIVALTGEKSPEE..RSKAWARAKVIVA T PQTIEND L LAGR...ISLEDVSLI Ib 370 380 390 400 410 420 430 RIG-I IF DE C H NTSKQHP Y NMIMFNYLDQKLGGS...SGPLPQVIG L T A SVGVGDAKNTDEALDYICKLC MDA5 II DE C H HTNKEAV Y NNIMRHYLMQKLKNNRLKKENKPVIPLPQILG L T A SPGVGGATKQAKAEEHILKLC LGP2 VV DE C H HTHKDTV Y NVIMSQYLELKLQR...AQPLPQVLG L T A SPGTGGASKLDGAINHVLQLC Dicer VF DE C H LAILDHP Y REIMKLCENCPSC...PRILG L T A SILNG.KCDPEELEEKIQKLE FANCM VI DE A H KALGNYA Y CQVVRELVKYTNH...FRILA L S A TPGSDIKAVQQVITNLLIGQI Hef VF DE A H RAVGNYA Y VFIAREYKRQAKN...PLVIG L T A SPGSTPEKIMEVINNLGIEHI II IIa III Ic 306 372 63 109 150 69 369 440 128 172 211 130 431 510 189 227 267 186 RIG-I MDA5 LGP2 Dicer FANCM Hef 440 450 460 470 480 490 500 ASLDASVIATVKHNLEELEQVVYKPQ.KFFRKVESRISDKFKYIIAQLMRDTESLAKRICKDLENLSQIQ ANLDAFTIKTVKENLDQLKNQIQEPC.KKFAIADATREDPFKEKLLEIMTRIQTYCQ...MSPMS ANLDTWCIMSPQNCCPQLQEHSQQPC.KQYNLCHRRSQDPFGDLLKKLMDQIHDHLE...MPELS KILKSN..AETATDLVVLDRYTSQPCEIVVDCGPFTDRSGLYERLLMELEEALNFIN...DCNISVHS ELRSEDSPDILTYSHERKVEKLIVPLGEELAAIQKTYIQILESFARSLIQRNVLMRR...DIPNLT EYRSENSPDVRPYVKGIRFEWVRVDLPEIYKEVRKLLREMLRDALKPLAETGLLES...SSPDIP 500 571 250 290 330 248 RIG-I MDA5 LGP2 Dicer FANCM Hef 510 520 530 540 550 560 570 NREFGTQKYEQWIVTVQKACMVFQMPDKDEESRICKALFLYTSHLRKYNDALIISEHARMKDALDYLKDF..DFGTQPYEQWAIQMEKKAAKKGNRKER...VCAEHLRKYNEALQINDTIRMIDAYTHLETF R.KFGTQMYEQQVVKLSEAAALAGLQEQR...VYALHLRRYNDALLIHDTVRAVDALAALQDF KERDSTLISKQILSDCRAVLVVLGPWCAD...KVAGMMVRELQKYIKHEQEELHRKFLLFTDTF K...YQIILARDQFRKNPSPNIVGIQQGI...IEGEFAICISLYHGYELLQQMGMRSLYFFLCGI K...KEVLRAGQIINEEMAKGNHDLRG...LLLYHAMALKLHHAIELLETQGLSALRAYIKKL 570 629 309 351 389 305 RIG-I MDA5 LGP2 Dicer FANCM Hef.... 580 590 600 FSNVRAAGFDEIE...Q.DLTQRFEEKLQELESVSRDPS... YNEEKDKKFAVIEDDSDEGGDDEYCDGDEDEDDLKKPLKLDETDRFLMTLFFENNKMLKRLAENPE... YHREHVTKTQILC...AERRLLALFDDRKNELAHLATHG... LRKIHALCEEHFSP...ASLDLKFVTPKVIKLLEILRKYKPYERQQFESVEWYNNRNQDNYVSWS MDGTKGMTRSKNE...LGRNE..D...FMKLYNHLECMFARTRSTSANGISAIQQGDKNKK YEEAKAGSTKAS...KEIFSDKRMKKAISLLVQAKEIG... 605 695 345 413 442 340 610 620 630 640 650 RIG-I..NENPKLEDLCFILQEEYHL...NPETIT.IL F VKT R ALVDALKNWIEGNPKLS...FLKPG MDA5..YENEKLTKLRNTIMEQYTR...TEESARGII F TKT R QSAYALSQWITENEKFAE..VGVKAH LGP2..PENPKLEMLEKILQRQFS...SSNSPRGII F TRT R QSAHSLLLWLQQQQGLQT..VDIRAQ Dicer DSEDDDEDEEIEEKEKPETNFP...SPFTNILCGII F VER R YTAVVLNRLIKEAGKQDPELAYISSN FANCM FVYSHPKLKKLEEVVIEHFKSWNAENTTEKKRDETRVMI F SSF R DSVQEIAEMLSQHQPI...IRVM Hef..LDHPKMDKLKEIIREQLQ...RK.QNSKIIV F TNY R ETAKKIVNELVKDG...IKAK 660 670 680 690 700 710 720 RIG-I ILT G RGKTNQNT...GMTLPA Q KCILDA F KASGDH N I L IA T SVAD EG I D IAQCNLVILYEYVGNVIKM MDA5 HLI G AGHSSEFK...PMTQNE Q KEVISK F RT.GKI N L L IA T TVAE EG L D IKECNIVIRYGLVTNEIAM LGP2 LLI G AGNSSQST...HMTQRD Q QEVIQK F QD.GTL N L L VA T SVAE EG L D IPHCNVVVRYGLLTNEISM Dicer FIT G HGIGKNQPRNKQMEAEFRK Q EEVLRK F RA.HET N L L IA T SIVE EG V D IPKCNLVVRFDLPTEYRSY FANCM TFV G HASGKSTK...GFTQKE Q LEVVKQ F RD.GGY N T L VS T CVGE EG L D IGEVDLIICFDSQKSPIRL Hef RFV G QASKENDR...GLSQRE Q KLILDE F AR.GEF N V L VA T SVGE EG L D VPEVDLVVFYEPVPSAIRS IVa IVb V Va Vb Vc IV 659 752 401 447 506 390 724 816 465 546 570 454 730 740 750 760 770 780 790 RIG-I I Q TR GR.G R ARGSK.CFLLTSNAGVIEKEQINMYKEKMMNDSILRLQTWDEAVFREKILHIQTHEKFIRD MDA5 V Q AR GR.A R ADESTYVLVAHSGSGVIEHETVNDFREKMMYKAIHCVQNMKPEEYAHKILELQMQSIMEKK LGP2 V Q AR GR.A R ADQSVYAFVATEGSRELKRELINEALETLMEQAVAAVQKMDQAEYQAKIRDLQQAALTKRA Dicer V Q SK GR.A R APISNYIMLADTDKIKSFEEDLKTYKAIEK... FANCM V Q RM GR TG R KRQGRIVIILSEGREERIYNQSQSNKRSIY... Hef I Q RR GR TG R HMPGRVIILMAKGTRDEAYYWSSRQKEKIM... VI 792 885 534 584 609 493 WWW.NATURE.COM/NATURE 3
800 810 820 830 840 850 SQEKPKPVPDKENK...KL LCRKCKALACYTADVRVIEECHYTVLGDAFKECF.VSR.PHPKPKQFSSF 856 MKTKRNIAKHYKNNPSLITF LCKNCSVLACSGEDIHVIEKMHHVNMTPEFKELY.IVRENKALQKKCADY 954 AQAAQRENQRQQFPVEHVQL LCINCMVAVGHGSDLRKVEGTHHVNVNPNFSNYYNVSRDPVVINKVFKDW 604 860 870 880 890 900 910 920 EKRAKIFCARQNCSHDWGIHVKYKTFEIPVIKIE.. SFVVEDIATGVQTLYSKWKDFHFEKIP. F D PAEM 923 QINGEIICK... C GQAWGTMMVHKGLDLPCLKIRNFVVVFKNNSTKKQ..YKKWVELPIT.FPNLDYSEC 1018 KPGGVISCR..NCGEVWGLQMIYKSVKLPVLKVR..SMLLETPQGRIQ..AKKWSRVPFS.VPDFDFLQH 667 SK... 925 CLFSDED... 1025 CAENLSDLSLD 678......... Supplementary Figure 3. Sequence alignment of RIG-I-like receptors. Multiple sequence alignment was carried out by using ClustalW, and illustrated with ESPript. Absolutely conserved residues in the helicase are shown as white text on a red background, while identical residues in the RD domain are shown as red text with a white background. Helicase motifs are boxed with colors according to domain localization as in Figure 1. Roman numerals indicate the position of conserved motifs found in RNA helicases and RNA helicase-like proteins. Red triangle indicates the residues involved in 5 strand RNA binding, whereas the residues involved in binding to 3 stand RNA are denoted with a blue triangle. Green triangle indicates the conserved residues involved in ATP analog binding. The secondary structure and residues numbers of RIG-I are marked on the top of sequence alignment. Accession numbers for each protein are as follows: RIG-I (NP_055129.2), MDA5 (NP_071451.2), LGP2 (NP_077024.2), Dicer (NP_085124.2), FANCM (NP_065988.1), and Hef (NP_579744.1). 4 WWW.NATURE.COM/NATURE
a b Domain 1 Domain 2 Domain 3 90 Domain 1 Domain 3 Domain 2 Supplementary Figure 4. Structural comparison of Hef helicase in the absence of nucleic acid and RIG-I helicase in the presence of dsrna. Overlay of Pyrococcus furiosus Hef helicase domain (PDB code 1WP9) with just the helicase domains of RIG-I and dsrna. The RIG-I domains are colored similar to Fig. 1. The view in panel b is rotated 90 about a horizontal axis from panel a. W W W. N A T U R E. C O M / N A T U R E 5
a Helicase-RD dsrna Data Helicase-RD dsrna Model Helicase-RD Data b Full-length RIG-I dsrna Data Full-length RIG-I dsrna Model Full-length RIG-I Data Log(I) Log(I) s (Å -1 ) s (Å -1 ) c Helicase-RD dsrna Helicase-RD d Full-length RIG-I dsrna Full-length RIG-I I*s 2 I*s 2 s (Å -1 ) s (Å -1 ) Supplementary Figure 5. SAXS analysis of helicase-rd and full-length RIG-I in the presence and absence of dsrna. Comparison of the experimental SAXS data of helicase-rd (a) and full-length RIG-I (b) in the presence and absence of dsrna to the calculated intensities based on the models presented in Fig. 4c-f using CRYSOL. (c and d) Kratky plots of helicase-rd (c) and full-length RIG-I (d) in the presence (blue) and absence (red) of 10 base pair dsrna. 6 WWW.NATURE.COM/NATURE
Supplementary Figure 6. Differential scanning fluorimetry of ligand (dsrna and ADP BeF 3 ) binding to helicase-rd (a) and full-length RIG-I (b). SYPRO Orange fluorescence is used as reporter of protein thermal stability. The apparent T m was determined by fitting a Boltzmann sigmoid to the fluorescence data. WWW.NATURE.COM/NATURE 7
Supplementary Table 1. Summary of the X-ray Crystallographic Analyses Selenomethionine Native Data collection Space group P6 5 22 P6 5 22 Cell dimensions a, b, c (Å) 174.9, 174.9, 110.9 175.4, 175.4, 109.2 α, β, γ ( ) 90.0, 90.0, 120.0 90.0, 90.0, 120.0 Resolution (Å) 30.0-3.2(3.47-3.2)* 30.0-2.9(3.1-2.9)* R merge 0.14(1.97) 0.18(2.83) R pim 0.03(0.34) 0.028(0.45) I/σI 21.1(2.4) 17.4(1.9) Completeness (%) 99.1(99.0) 99.8(99.8) Redundancy 37.9(33.1) 39.4(40.0) Refinement Resolution (Å) 18.06-2.90 No. reflections 22245 R work/ R free 0.199/0.287 No. atoms Protein 5196 Ligand/ion 544 B-factors (Å 2 ) a Protein 117.1 Ligand/ion 126.3 R.m.s deviations Bond lengths (Å) 0.014 Bond angles (º) 1.266 * Highest resolution shell is shown in parenthesis. a. The B-factor from coordinate refinement is consistent with estimates from the Wilson plot analysis of the data. 8 WWW.NATURE.COM/NATURE
Supplementary Table 2. SAXS data parameters and quality metrics. Helicase-RD Helicase-RD +dsrna Full-Length Full-Length +dsrna R g (Å) a 37.8±0.1 27.7±0.1 41.7±0.1 39.4±0.1 D max (Å) b 135 85 180 120 NSD c 0.63±0.06 0.49±0.01 0.97±0.04 0.57±0.03 χ 2 d - 4.25-6.73 a Calculated using PRIMUS b Determined using GNOM and analyzed using MATLAB c Agreement between ab initio models calculated using SUPCOMB20 d Consistency between ab initio model and crystal structure docking. Numbers were calculated using CRYSOL WWW.NATURE.COM/NATURE 9