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/ Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300. Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300. mBio 2021 imex curation Davis DA., Bulut H., Shrestha P., Yaparla A., Jaeger HK., Hattori SI., Wingfield PT., Mieyal JJ., Mitsuya H., Yarchoan R. Virus - Publications including interactions involving viral proteins Coronavirus - Interactions investigated in the context of Coronavirus Figure S7 (A-D) refers to "assays carried out in figure 6 A/B" - only figure 6 is a model only. dd162w@nih.gov Only protein-protein interactions imex curation in vitro In vitro solution sedimentati cosedimentation in solution solution sedimentati predetermined predetermined participant predetermined Davis DA., Bulut H., Shrestha P., Yaparla A., Jaeger HK., Hattori SI., Wingfield PT., Mieyal JJ., Mitsuya H., Yarchoan R. Virus - Publications including interactions involving viral proteins Figure S7 (A-D) refers to "assays carried out in figure 6 A/B" - only figure 6 is a model only. dd162w@nih.gov Accepted 2021-OCT-18 AT 12:38 BST AT 12:38 BST by KALPANAP mBio 2021 Only protein-protein interactions imex curation imex curation Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300. Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300. mBio 2021 imex curation Davis DA., Bulut H., Shrestha P., Yaparla A., Jaeger HK., Hattori SI., Wingfield PT., Mieyal JJ., Mitsuya H., Yarchoan R. Virus - Publications including interactions involving viral proteins Coronavirus - Interactions investigated in the context of Coronavirus Figure S7 (A-D) refers to "assays carried out in figure 6 A/B" - only figure 6 is a model only. dd162w@nih.gov Only protein-protein interactions imex curation in vitro In vitro oxidoreduct assay oxidoreductase assay oxidoreduct assay ms participant Identification by mass spectrometry ms participant Davis DA., Bulut H., Shrestha P., Yaparla A., Jaeger HK., Hattori SI., Wingfield PT., Mieyal JJ., Mitsuya H., Yarchoan R. Virus - Publications including interactions involving viral proteins Figure S7 (A-D) refers to "assays carried out in figure 6 A/B" - only figure 6 is a model only. dd162w@nih.gov Accepted 2021-OCT-18 AT 12:38 BST AT 12:38 BST by KALPANAP mBio 2021 Only protein-protein interactions imex curation imex curation Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300. Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300. mBio 2021 imex curation Davis DA., Bulut H., Shrestha P., Yaparla A., Jaeger HK., Hattori SI., Wingfield PT., Mieyal JJ., Mitsuya H., Yarchoan R. Virus - Publications including interactions involving viral proteins Coronavirus - Interactions investigated in the context of Coronavirus Figure S7 (A-D) refers to "assays carried out in figure 6 A/B" - only figure 6 is a model only. dd162w@nih.gov Only protein-protein interactions imex curation in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column experimental particp experimental participant identification experimental particp Davis DA., Bulut H., Shrestha P., Yaparla A., Jaeger HK., Hattori SI., Wingfield PT., Mieyal JJ., Mitsuya H., Yarchoan R. Virus - Publications including interactions involving viral proteins Figure S7 (A-D) refers to "assays carried out in figure 6 A/B" - only figure 6 is a model only. dd162w@nih.gov Accepted 2021-OCT-18 AT 12:38 BST AT 12:38 BST by KALPANAP mBio 2021 Only protein-protein interactions imex curation imex curation Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300. Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300. mBio 2021 imex curation Davis DA., Bulut H., Shrestha P., Yaparla A., Jaeger HK., Hattori SI., Wingfield PT., Mieyal JJ., Mitsuya H., Yarchoan R. Virus - Publications including interactions involving viral proteins Coronavirus - Interactions investigated in the context of Coronavirus Figure S7 (A-D) refers to "assays carried out in figure 6 A/B" - only figure 6 is a model only. dd162w@nih.gov Only protein-protein interactions imex curation in vitro In vitro protease assay protease assay predetermined predetermined participant predetermined Davis DA., Bulut H., Shrestha P., Yaparla A., Jaeger HK., Hattori SI., Wingfield PT., Mieyal JJ., Mitsuya H., Yarchoan R. Virus - Publications including interactions involving viral proteins Figure S7 (A-D) refers to "assays carried out in figure 6 A/B" - only figure 6 is a model only. The peptide substrate is cleaved at the Gln-p-nitroaniline bond to release free p-nitroaniline, resulting in an increase in absorbance. dd162w@nih.gov Accepted 2021-OCT-18 AT 12:38 BST AT 12:38 BST by KALPANAP mBio 2021 Only protein-protein interactions imex curation imex curation Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300. Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300. mBio 2021 imex curation Davis DA., Bulut H., Shrestha P., Yaparla A., Jaeger HK., Hattori SI., Wingfield PT., Mieyal JJ., Mitsuya H., Yarchoan R. Virus - Publications including interactions involving viral proteins Coronavirus - Interactions investigated in the context of Coronavirus Figure S7 (A-D) refers to "assays carried out in figure 6 A/B" - only figure 6 is a model only. dd162w@nih.gov Only protein-protein interactions imex curation in vitro In vitro ms interact detect detection by mass spectrometry ms interact detect experimental particp experimental participant identification experimental particp Davis DA., Bulut H., Shrestha P., Yaparla A., Jaeger HK., Hattori SI., Wingfield PT., Mieyal JJ., Mitsuya H., Yarchoan R. Virus - Publications including interactions involving viral proteins Figure S7 (A-D) refers to "assays carried out in figure 6 A/B" - only figure 6 is a model only. dd162w@nih.gov Accepted 2021-OCT-18 AT 12:38 BST AT 12:38 BST by KALPANAP mBio 2021 Only protein-protein interactions imex curation imex curation glutathione glutathione gamma-L-Glutamyl-L-cysteinyl-glycine 5-L-Glutamyl-L-cysteinylglycine Glutathione Glutathione-SH GSH N-(N-gamma-L-Glutamyl-L-cysteinyl)glycine Reduced glutathione L-gamma-glutamyl-L-cysteinylglycine small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) glutathione disulfid glutathione disulfide Glutathione disulfide glutathione disulphide GSSG oxidised glutathione Oxidized glutathione OXIDIZED GLUTATHIONE DISULFIDE Oxiglutatione (2S,2'S)-5,5'-[disulfanediylbis({(2R)-3-[(carboxymethyl)amino]-3-oxopropane-1,2-diyl}imino)]bis(2-amino-5-oxopentanoic acid) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) YPZRWBKMTBYPTK-BJDJZHNGSA-N YPZRWBKMTBYPTK-BJDJZHNGSA-N glrx1_human Glutaredoxin-1 GLRX GRX Thioltransferase-1 protein protein human Homo sapiens Human MAQEFVNCKIQPGKVVVFIKPTCPYCRRAQEILSQLPIKQGLLEFVDITATNHTNEIQDYLQQLTGARTVPRVFIGKDCIGGCSDLVSLQQSGELLTRLKQIGALQ BB86FED55967EBE2 hydrogen peroxide hydrogen peroxide [OH(OH)] dihydrogen dioxide H2O2 HOOH Hydrogen peroxide HYDROGEN PEROXIDE Oxydol bis(hydridooxygen)(O--O) dihydrogen peroxide dihydrogen(peroxide) dioxidane hydrogen peroxide small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) MHAJPDPJQMAIIY-UHFFFAOYSA-N MHAJPDPJQMAIIY-UHFFFAOYSA-N tsavlq peptide peptide polypeptide oligopeptide oligopeptide polypeptide chemical synthesis Chemical synthesis (Chemical synthesis) TSAVLQ d-1,4-dithiothreitol D-1,4-dithiothreitol (2S,3S)-1,4-dimercaptobutane-2,3-diol D-DTT D-threo-1,4-dimercapto-2,3-butanediol (2S,3S)-1,4-disulfanylbutane-2,3-diol small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) p0dtd1-pro_0000449623 3C-like proteinase nsp5 rep ORF1ab polyprotein 1a-1b protein protein SARS-CoV-2 SARS-CoV-2 SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 2019-nCoV SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDVVYCPRHVICTSEDMLNPNYEDLLIRKSNHNFLVQAGNVQLRVIGHSMQNCVLKLKVDTANPKTPKYKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNFTIKGSFLNGSCGSVGFNIDYDCVSFCYMHHMELPTGVHAGTDLEGNFYGPFVDRQTAQAAGTDTTITVNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDILGPLSAQTGIAVLDMCASLKELLQNGMNGRTILGSALLEDEFTPFDVVRQCSGVTFQ 3264 3569 tcep TCEP 3,3',3''-phosphinidynetrispropanoic acid tris(2-carboxyethyl)phosphine 3,3',3"-phosphanetriyltripropanoic acid small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) rep-tsavlq-1 4 12 enzyme enzyme neutral component neutral component purified purified P0DTD1-PRO_0000449623:p.Cys300Ser mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain C S Tue Oct 05 19:08:33 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys300Ser' ecoli Escherichia coli (strain K12) 10 enzyme target enzyme target substrate substrate neutral component neutral component para-nitro-aniline modgln modified L-glutamine residue ModGln c-term range C-terminal range c-term range c-term range C-terminal range c-term range chemical synthesis Chemical synthesis (Chemical synthesis) protein cleavage protein cleavage 1A, 3D, 4D/E, 5A MPro at ~~1.2 uM. rep-tsavlq-2 4 7 inhibitor inhibitor neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 10 enzyme target enzyme target substrate substrate neutral component neutral component para-nitro-aniline modgln modified L-glutamine residue ModGln c-term range C-terminal range c-term range c-term range C-terminal range c-term range chemical synthesis Chemical synthesis (Chemical synthesis) 12 enzyme enzyme neutral component neutral component purified purified P0DTD1-PRO_0000449623:p.Cys300Ser mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain C S Tue Oct 05 18:55:18 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys300Ser' ecoli Escherichia coli (strain K12) protein cleavage protein cleavage 1A, 5A MPro at 1.2 uM (mainly monomeric). GSH had little effect at this concentration. When Mpro concentration was increased to 18 uM (mainly dimeric form), it was resistant to GSSG inhibition. glutathione disulfid-rep-1 5 7 unspecified role unspecified role neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 12 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified P0DTD1-PRO_0000449623:p.Cys300Ser mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain C S Tue Oct 05 18:49:43 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys300Ser' glutathionylated residue modres protein modification ModRes undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined resulting-ptm resulting-ptm resulting ptm resulting-ptm ecoli Escherichia coli (strain K12) redox reaction oxidoreductase activity electron transfer reaction redox reaction 1C-E, Supp. figure 7 GSSG glutathionylates SARS-CoV-2 Mpro at low Mpro concentrations when Mpro is largely monomeric. Treatment of 1.2 uM Mpro with 2 mM GSSG led to ~ 45% monoglutathionylation; treatment with 10 mM GSSG led to mono- (11%), di- (50%), and triglutathionylation (35%), with less than 4% of Mpro remaining unmodified. glutathione disulfid-rep-2 5 11 unspecified role unspecified role neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 12 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified glutathionylated residue disulfide conjugated residue disulfide conjugated residue S-thiolation undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) redox reaction oxidoreductase activity electron transfer reaction redox reaction 1F d_1,4_dithiothreitol-rep-1 4 10 enzyme target enzyme target substrate substrate neutral component neutral component para-nitro-aniline modgln modified L-glutamine residue ModGln c-term range C-terminal range c-term range c-term range C-terminal range c-term range chemical synthesis Chemical synthesis (Chemical synthesis) 11 stimulator stimulator neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 12 enzyme enzyme neutral component neutral component purified purified P0DTD1-PRO_0000449623:p.Cys300Ser mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain C S Tue Oct 05 19:25:31 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys300Ser' glutathionylated residue disulfide conjugated residue disulfide conjugated residue S-thiolation undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) protein cleavage protein cleavage 1G, 5B Glutathionylated Mpro had less than 5% of the activity of unmodified Mpro; following the addition of 10 mM DTT, the activity was fully restored. Mpro at 1uM. rep-284 3 12 predetermined predetermined participant predetermined ms participant Identification by mass spectrometry ms participant experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) direct interaction direct interaction 2A-F, 3 A-C, Supp. figure 3 Native Mpro was in equilibrium between monomeric and dimeric forms: glutathionylated Mpro behaved as a monomer. rep-285 1 12 predetermined predetermined participant predetermined ms participant Identification by mass spectrometry ms participant predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) direct interaction direct interaction 2G Average M. wt =63kDa (dimeric) rep-286 1 12 unspecified role unspecified role neutral component neutral component purified purified glutathionylated residue disulfide conjugated residue disulfide conjugated residue S-thiolation undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction 2H Glutathionylated Mpro behaved almost completely as a monomer:Average M. Wt = 37kDa. only entered as a dimer as they give a Kd value rep-tcep-1 3 13 unspecified role unspecified role neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 12 predetermined predetermined participant predetermined ms participant Identification by mass spectrometry ms participant experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified glutathionylated residue disulfide conjugated residue disulfide conjugated residue S-thiolation undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) redox reaction oxidoreductase activity electron transfer reaction redox reaction Supp. figure 3 TCEP removes the glutathione moiety causing an increase in the dimer peak and decrease in the monomer peak. rep-tcep-2 4 10 enzyme target enzyme target substrate substrate neutral component neutral component para-nitro-aniline modgln modified L-glutamine residue ModGln c-term range C-terminal range c-term range c-term range C-terminal range c-term range chemical synthesis Chemical synthesis (Chemical synthesis) 13 stimulator stimulator neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 12 enzyme enzyme neutral component neutral component purified purified glutathionylated residue disulfide conjugated residue disulfide conjugated residue S-thiolation undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) protein cleavage protein cleavage 3D Treatment with TCEP to remove the glutathione moiety, resulted in a significant increase in activity. glutathione-rep-1 2 8 enzyme enzyme neutral component neutral component purified purified region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. 6 cofactor cofactor coenzyme neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 12 predetermined predetermined participant predetermined ms participant Identification by mass spectrometry ms participant ms participant Identification by mass spectrometry ms participant enzyme target enzyme target substrate substrate neutral component neutral component purified purified glutathionylated residue disulfide conjugated residue disulfide conjugated residue S-thiolation undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) redox reaction oxidoreductase activity electron transfer reaction redox reaction 4 A-C, Supp. figure 4 A-D Incubation of glutathionylated Mpro with Grx and GSH resulted a decrease in glutathionylated Mpro. glutathione-rep-2 4 8 stimulator stimulator neutral component neutral component purified purified region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. 10 enzyme target enzyme target substrate substrate neutral component neutral component para-nitro-aniline modgln modified L-glutamine residue ModGln c-term range C-terminal range c-term range c-term range C-terminal range c-term range chemical synthesis Chemical synthesis (Chemical synthesis) 6 cofactor cofactor coenzyme neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 12 enzyme enzyme neutral component neutral component purified purified glutathionylated residue disulfide conjugated residue disulfide conjugated residue S-thiolation undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) protein cleavage protein cleavage 4 D/E, Supp. figure 4E Incubation of glutathionylated Mpro with Grx in the presence of GSH led to a significant increase in protease activity. rep-tcep-3 5 13 unspecified role unspecified role neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 12 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified glutathionylated residue disulfide conjugated residue disulfide conjugated residue S-thiolation undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined cys300 modcys modified L-cysteine residue ModCys certain certain sequence position certain certain certain sequence position certain cys156 modcys modified L-cysteine residue ModCys certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) redox reaction oxidoreductase activity electron transfer reaction redox reaction Table 1,2 Figure S6 Peptide digests containing glutathionylated forms of mpro were treated with TCEP to remove disulfide-bound glutathione. Subsequently, masses for predicted glutathionylated peptides were no longer detected. The majority of the monoglutathionylation was occurring at Cys300. glutathione-rep-3 5 9 electron acceptor electron acceptor neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 6 electron donor electron donor neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 12 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified P0DTD1-PRO_0000449623:p.Cys300Ser mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain C S Tue Oct 05 19:10:47 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys300Ser' glutathionylated residue modcys modified L-cysteine residue ModCys undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined resulting-ptm resulting-ptm resulting ptm resulting-ptm ecoli Escherichia coli (strain K12) redox reaction oxidoreductase activity electron transfer reaction redox reaction Supp. figure 8