IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ http://www.ebi.ac.uk/ gao-2013-2 Structure-Function Analysis of STING Activation by c[G(2',5')pA(3',5')p] and Targeting by Antiviral DMXAA. in vitro In vitro itc isothermal titration calorimetry ITC ITC itc predetermined predetermined participant predetermined Gao P., Ascano M., Zillinger T., Wang W., Dai P., Serganov A.A., Gaffney B.L., Shuman S., Jones R.A., Deng L., Hartmann G., Barchet W., Tuschl T., Patel D.J. winfried.barchet@ukb.uni-bonn.de, ttuschl@mail.rockefeller.edu, pateld@mskcc.org Cell (0092-8674) 2013 gao-2013-1 Structure-Function Analysis of STING Activation by c[G(2',5')pA(3',5')p] and Targeting by Antiviral DMXAA. in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Gao P., Ascano M., Zillinger T., Wang W., Dai P., Serganov A.A., Gaffney B.L., Shuman S., Jones R.A., Deng L., Hartmann G., Barchet W., Tuschl T., Patel D.J. winfried.barchet@ukb.uni-bonn.de, ttuschl@mail.rockefeller.edu, pateld@mskcc.org Cell (0092-8674) 2013 sting_human Stimulator of interferon genes protein STING1 ERIS Endoplasmic reticulum interferon stimulator MITA Mediator of IRF3 activation TMEM173 Transmembrane protein 173 protein protein human Homo sapiens Human MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS cg(2'5')_pa(3'5')p small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) c-gmp-amp c-GMP-AMP 3',5'-cyclic GMP-AMP cyclic GMP-AMP 2-amino-9-[(2R,3R,3aS,7aR,9R,10R,10aS,14aR)-9-(6-amino-9H-purin-9-yl)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecin-2-yl]-1,9-dihydro-6H-purin-6-one small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) RFCBNSCSPXMEBK-INFSMZHSSA-N cg(2'5')_pa(2'5')p small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) sting_mouse Stimulator of interferon genes protein Sting1 Endoplasmic reticulum interferon stimulator Eris Mediator of IRF3 activation Mita Mpys Tmem173 Transmembrane protein 173 protein protein mouse Mus musculus Mouse MPYSNLHPAIPRPRGHRSKYVALIFLVASLMILWVAKDPPNHTLKYLALHLASHELGLLLKNLCCLAEELCHVQSRYQGSYWKAVRACLGCPIHCMAMILLSSYFYFLQNTADIYLSWMFGLLVLYKSLSMLLGLQSLTPAEVSAVCEEKKLNVAHGLAWSYYIGYLRLILPGLQARIRMFNQLHNNMLSGAGSRRLYILFPLDCGVPDNLSVVDPNIRFRDMLPQQNIDRAGIKNRVYSNSVYEILENGQPAGVCILEYATPLQTLFAMSQDAKAGFSREDRLEQAKLFCRTLEEILEDVPESRNNCRLIVYQEPTDGNSFSLSQEVLRHIRQEEKEEVTMNAPMTSVAPPPSVLSQEPRLLISGMDQPLPLRTDLI dmxaa (5,6-dimethyl-9-oxo-9H-xanthen-4-yl)acetic acid small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) c_gmp_amp-cg_2_5__ 1446325 n/a 1446327 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Q86WV6:p.Arg238Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false Q86WV6:p.Tyr240Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false Q86WV6:p.Asn242Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false Q86WV6:p.Glu260Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false Q86WV6:p.Ser162Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false Q86WV6:p.Thr263Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false Q86WV6:p.His232Arg mutation increasing strength mutation increasing interaction strength mutation increasing strength certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 n/a 1446328 unspecified role unspecified role neutral component neutral component Stoichiometry: 1.0 direct interaction direct interaction false false false 1446325 Fig. 4A, 4B, 4C, Supp. Table 3, 4 CHEBI:75947 increases activity of Q86WV6 c_gmp_amp-c_gmp_amp_ 1446325 n/a 1446329 unspecified role unspecified role neutral component neutral component Stoichiometry: 1.0 n/a 1446327 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Q86WV6:p.His232Arg mutation increasing strength mutation increasing interaction strength mutation increasing strength certain certain sequence position certain certain certain sequence position certain false Q86WV6:p.[Gly230Ala;His232Arg] mutation increasing strength mutation increasing interaction strength mutation increasing strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false 1446325 The 3′,5′-containing linkage isomers of cGAMP exhibited increased binding affinity by about 4-fold for STING variants with R232 compared to H232. Endothermic titration patterns were observed for all binding curves, except for an exothermic titration pattern for c[G(3′,5′)pA(3′,5′)p] with hSTINGR232 Fig. 4A, C, D, Supp. Table 3 CHEBI:71580 increases activity of Q86WV6 cg_2_5__-tmem173 1446325 n/a 1446330 unspecified role unspecified role neutral component neutral component Stoichiometry: 1.0 n/a 1446327 unspecified role unspecified role neutral component neutral component Q86WV6:p.[Gly230Ala;His232Arg] mutation increasing strength mutation increasing interaction strength mutation increasing strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Q86WV6:p.His232Arg mutation increasing strength mutation increasing interaction strength mutation increasing strength certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false 1446325 The 2′,5′-containing linkage isomers of cGAMP exhibited increased binding affinity by aboutan order of magnitude for STING variants with R232 compared to H232. Endothermic titration patterns were observed for all binding curves, except for an exothermic titration pattern for c[G(3′,5′)pA(3′,5′)p] with hSTINGR232 Fig. 4A, C, D, Supp. Table 3 CHEBI:75948 increases activity of Q86WV6 c_gmp_amp-tmem173-2 1446325 n/a 1446331 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Q3TBT3:p.Arg231Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 n/a 1446329 unspecified role unspecified role neutral component neutral component Stoichiometry: 1.0 direct interaction direct interaction false false false 1446325 Fig. 4F, Supp Table 3 CHEBI:71580 increases activity of Q3TBT3 c_gmp_amp_2-cg_2_5__ 1446325 n/a 1446331 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Q3TBT3:p.Arg231Ala mutation mutation certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 n/a 1446328 unspecified role unspecified role neutral component neutral component Stoichiometry: 1.0 direct interaction direct interaction false false false 1446325 Fig. 4F Supp Table 3 CHEBI:75947 increases activity of Q3TBT3 cg_2_5__-tmem173-1 1446325 n/a 1446331 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 n/a 1446330 unspecified role unspecified role neutral component neutral component Stoichiometry: 1.0 direct interaction direct interaction false false false 1446325 Fig. 4E Supp Table 3 CHEBI:75948 increases activity of Q3TBT3 dmxaa-tmem173 1446325 n/a 1446331 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1446332 unspecified role unspecified role neutral component neutral component direct interaction direct interaction false false false Fig. 7E CHEBI:75934 increases activity of Q3TBT3 dmxaa-tmem173-1 1446325 n/a 1446332 unspecified role unspecified role neutral component neutral component n/a 1446327 unspecified role unspecified role neutral component neutral component Q86WV6:p.His232Arg mutation mutation certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Fig. 7F, 7G cg_2_5_-tmem173 1446326 n/a 1446328 unspecified role unspecified role neutral component neutral component Stoichiometry: 1.0 n/a 1446327 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false 2.25A Rworking = 0.19 Rfree = 0.22 Supp. Table 1 CHEBI:75947 increases activity of Q86WV6 c_gmp_amp-cg_2_5_ 1446326 n/a 1446330 unspecified role unspecified role neutral component neutral component Stoichiometry: 1.0 n/a 1446327 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false 1.89A Rworking = 0.18 Rfree = 0.2 Supp. Table 1 CHEBI:75948 increases activity of Q86WV6 c_gmp_amp_2_-cg_2_5_ 1446326 n/a 1446331 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 n/a 1446328 unspecified role unspecified role neutral component neutral component Stoichiometry: 1.0 direct interaction direct interaction false false false Supp Table 2 1.77A Rworking: 19.2%, Free R-factor: 21.2% CHEBI:75947 increases activity of Q3TBT3 c_gmp_amp-tmem173-1 1446326 n/a 1446331 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 n/a 1446329 unspecified role unspecified role neutral component neutral component Stoichiometry: 1.0 direct interaction direct interaction false false false Supp Table 2 2.07A Rworking: 0.18 Rfree = 0.24 CHEBI:71580 increases activity of Q3TBT3 _5_6_dimethy-tmem173 1446326 n/a 1446331 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 n/a 1446332 unspecified role unspecified role neutral component neutral component Stoichiometry: 2.0 direct interaction direct interaction false false false Supp Table 2 2.43A Rworking = 0.18 Rfree = 0.21