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/ New targets for drug design: Importance of nsp14/nsp10 complex formation for the 3'-5' exoribonucleolytic activity on SARS-CoV-2. New targets for drug design: Importance of nsp14/nsp10 complex formation for the 3'-5' exoribonucleolytic activity on SARS-CoV-2. The FEBS journal 2021 imex curation Saramago M., Bárria C., Costa VG., Souza CS., Viegas SC., Domingues S., Lousa D., Soares CM., Arraiano CM., Matos RG. Virus - Publications including interactions involving viral proteins Coronavirus - Interactions investigated in the context of Coronavirus Figure 1A; under assay conditions I see no substantial evidence of higher order "reminiscent" oligomers, despite text and "data-not shown". margaridasaramago@itqb.unl.pt, cecilia@itqb.unl.pt, rmatos@itqb.unl.pt 24-May-2021: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro ribonuclease assay ribonuclease assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Saramago M., Bárria C., Costa VG., Souza CS., Viegas SC., Domingues S., Lousa D., Soares CM., Arraiano CM., Matos RG. Virus - Publications including interactions involving viral proteins Figure 1A; under assay conditions I see no substantial evidence of higher order "reminiscent" oligomers, despite text and "data-not shown". margaridasaramago@itqb.unl.pt, cecilia@itqb.unl.pt, rmatos@itqb.unl.pt Accepted 2021-APR-04 AT 17:02 BST AT 17:02 BST by KALPANAP The FEBS journal 2021 Only protein-protein interactions imex curation imex curation New targets for drug design: Importance of nsp14/nsp10 complex formation for the 3'-5' exoribonucleolytic activity on SARS-CoV-2. New targets for drug design: Importance of nsp14/nsp10 complex formation for the 3'-5' exoribonucleolytic activity on SARS-CoV-2. The FEBS journal 2021 imex curation Saramago M., Bárria C., Costa VG., Souza CS., Viegas SC., Domingues S., Lousa D., Soares CM., Arraiano CM., Matos RG. Virus - Publications including interactions involving viral proteins Coronavirus - Interactions investigated in the context of Coronavirus Figure 1A; under assay conditions I see no substantial evidence of higher order "reminiscent" oligomers, despite text and "data-not shown". margaridasaramago@itqb.unl.pt, cecilia@itqb.unl.pt, rmatos@itqb.unl.pt 24-May-2021: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Saramago M., Bárria C., Costa VG., Souza CS., Viegas SC., Domingues S., Lousa D., Soares CM., Arraiano CM., Matos RG. Virus - Publications including interactions involving viral proteins Figure 1A; under assay conditions I see no substantial evidence of higher order "reminiscent" oligomers, despite text and "data-not shown". margaridasaramago@itqb.unl.pt, cecilia@itqb.unl.pt, rmatos@itqb.unl.pt Accepted 2021-APR-04 AT 17:02 BST AT 17:02 BST by KALPANAP The FEBS journal 2021 Only protein-protein interactions imex curation imex curation New targets for drug design: Importance of nsp14/nsp10 complex formation for the 3'-5' exoribonucleolytic activity on SARS-CoV-2. New targets for drug design: Importance of nsp14/nsp10 complex formation for the 3'-5' exoribonucleolytic activity on SARS-CoV-2. The FEBS journal 2021 imex curation Saramago M., Bárria C., Costa VG., Souza CS., Viegas SC., Domingues S., Lousa D., Soares CM., Arraiano CM., Matos RG. Virus - Publications including interactions involving viral proteins Coronavirus - Interactions investigated in the context of Coronavirus Figure 1A; under assay conditions I see no substantial evidence of higher order "reminiscent" oligomers, despite text and "data-not shown". margaridasaramago@itqb.unl.pt, cecilia@itqb.unl.pt, rmatos@itqb.unl.pt 24-May-2021: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro methyltransferase as methyltransferase assay methyltransferase as weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Saramago M., Bárria C., Costa VG., Souza CS., Viegas SC., Domingues S., Lousa D., Soares CM., Arraiano CM., Matos RG. Virus - Publications including interactions involving viral proteins Figure 1A; under assay conditions I see no substantial evidence of higher order "reminiscent" oligomers, despite text and "data-not shown". Interactions (the conversion of G*pppRNA to m7G*pppRNA) were detected by relative movement of radioactive species via TLC. margaridasaramago@itqb.unl.pt, cecilia@itqb.unl.pt, rmatos@itqb.unl.pt Accepted 2021-APR-04 AT 17:02 BST AT 17:02 BST by KALPANAP The FEBS journal 2021 Only protein-protein interactions imex curation imex curation New targets for drug design: Importance of nsp14/nsp10 complex formation for the 3'-5' exoribonucleolytic activity on SARS-CoV-2. New targets for drug design: Importance of nsp14/nsp10 complex formation for the 3'-5' exoribonucleolytic activity on SARS-CoV-2. The FEBS journal 2021 imex curation Saramago M., Bárria C., Costa VG., Souza CS., Viegas SC., Domingues S., Lousa D., Soares CM., Arraiano CM., Matos RG. Virus - Publications including interactions involving viral proteins Coronavirus - Interactions investigated in the context of Coronavirus Figure 1A; under assay conditions I see no substantial evidence of higher order "reminiscent" oligomers, despite text and "data-not shown". margaridasaramago@itqb.unl.pt, cecilia@itqb.unl.pt, rmatos@itqb.unl.pt 24-May-2021: Contacted by IntAct-Help. Only protein-protein interactions imex curation ecoli Escherichia coli (strain K12) molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined Saramago M., Bárria C., Costa VG., Souza CS., Viegas SC., Domingues S., Lousa D., Soares CM., Arraiano CM., Matos RG. Virus - Publications including interactions involving viral proteins Figure 1A; under assay conditions I see no substantial evidence of higher order "reminiscent" oligomers, despite text and "data-not shown". margaridasaramago@itqb.unl.pt, cecilia@itqb.unl.pt, rmatos@itqb.unl.pt Accepted 2021-APR-04 AT 17:02 BST AT 17:02 BST by KALPANAP The FEBS journal 2021 Only protein-protein interactions imex curation imex curation ds_h4_rna ds rna ds rna double stranded ribonucleic acid chemical synthesis Chemical synthesis (Chemical synthesis) UGACGGCCCGGAAAACCGGGCC p0dtd1-pro_0000449628 Non-structural protein 10 rep ORF1ab polyprotein 1a-1b protein protein SARS-CoV-2 SARS-CoV-2 SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 2019-nCoV AGNATEVPANSTVLSFCAFAVDAAKAYKDYLASGGQPITNCVKMLCTHTGTGQAITVTPEANMDQESFGGASCCLYCRCHIDHPNPKGFCDLKGKYVQIPTTCANDPVGFTLKNTVCTVCGMWKGYGCSCDQLREPMLQ 4254 4392 p0dtd1-pro_0000449631 Proofreading exoribonuclease nsp14 rep ORF1ab polyprotein 1a-1b protein protein SARS-CoV-2 SARS-CoV-2 SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 2019-nCoV AENVTGLFKDCSKVITGLHPTQAPTHLSVDTKFKTEGLCVDIPGIPKDMTYRRLISMMGFKMNYQVNGYPNMFITREEAIRHVRAWIGFDVEGCHATREAVGTNLPLQLGFSTGVNLVAVPTGYVDTPNNTDFSRVSAKPPPGDQFKHLIPLMYKGLPWNVVRIKIVQMLSDTLKNLSDRVVFVLWAHGFELTSMKYFVKIGPERTCCLCDRRATCFSTASDTYACWHHSIGFDYVYNPFMIDVQQWGFTGNLQSNHDLYCQVHGNAHVASCDAIMTRCLAVHECFVKRVDWTIEYPIIGDELKINAACRKVQHMVVKAALLADKFPVLHDIGNPKAIKCVPQADVEWKFYDAQPCSDKAYKIEELFYSYATHSDKFTDGVCLFWNCNVDRYPANSIVCRFDTRVLSNLNLPGCDGGSLYVNKHAFHTPAFDKSAFVNLKQLPFFYYSDSPCESHGKQVVSDIDYVPLKSATCITRCNLGGAVCRHHANEYRLYLDAYNMMISAGFSLWVYKQFDTYNLWNTFTRLQ 5926 6452 30mer_gppp-rna rna ribonucleic acid RNA rna in vitro In vitro GNNNNNNNNNNNNNNNNNNNNNNNNNNNNN ds_h4_rna-rep-2 1 5 enzyme target enzyme target substrate substrate neutral component neutral component 5' 32p radiolabel 32p radiolabel P32 32P 32P P32 certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) 6 stimulator stimulator neutral component neutral component purified purified P0DTD1-PRO_0000449628:p.His80Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain H A Thu Apr 22 11:52:51 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.His80Ala' P0DTD1-PRO_0000449628:p.Phe19Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain F A Thu Apr 22 13:38:07 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe19Ala' P0DTD1-PRO_0000449628:p.Gly69Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain G A Thu Apr 22 13:25:48 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gly69Ala' P0DTD1-PRO_0000449628:p.Tyr96Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain Y A Thu Apr 22 10:27:42 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Tyr96Ala' n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range P0DTD1-PRO_0000449628:p.Ser72Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain S A ecoli Escherichia coli (strain K12) 7 enzyme enzyme neutral component neutral component purified purified n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) rna cleavage rna cleavage 1C, 1E, 2, 4D, 5C Co-purifying nsp14 and nsp10 proteins substantially increases nsp14 ExoNase activity. MnCl2 Mgcl2 Addition of ZnCl2 and CoCl2 caused a slight increase in ExoN activity. EDTA (CHEBI:42191 ) Mutations of nsp10 (with the exception of S72A) reduced ExoNase activity to that of nsp14 alone. The S72A mutation further decreased activity. ds_h4_rna-rep-1 1 5 enzyme target enzyme target substrate substrate neutral component neutral component 5' 32p radiolabel 32p radiolabel P32 32P 32P P32 certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) 7 enzyme enzyme neutral component neutral component purified purified n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) rna cleavage rna cleavage 1C, 1E, 4D, 5C nsp14 alone exhibits exoribonucleolytic activity; however low activity was seen. rep-rep-147 2 6 unspecified role unspecified role prey prey purified purified n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range P0DTD1-PRO_0000449628:p.Phe19Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain F A Thu Apr 22 10:50:04 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe19Ala' P0DTD1-PRO_0000449628:p.Ser72Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain S A Thu Apr 22 13:30:51 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser72Ala' P0DTD1-PRO_0000449628:p.Gly69Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain G A Thu Apr 22 12:28:37 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gly69Ala' P0DTD1-PRO_0000449628:p.His80Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain H A Thu Apr 22 10:16:12 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.His80Ala' P0DTD1-PRO_0000449628:p.Tyr96Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain Y A Thu Apr 22 12:26:53 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Tyr96Ala' ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role bait bait purified purified n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) direct interaction direct interaction 1D, 4C As both proteins are unstable, it was not possible to obtain a good fit of the data in order to determine the binding kinetics and dissociation constants. rep-rep-148 4 6 unspecified role unspecified role neutral component neutral component purified purified n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 7 unspecified role unspecified role neutral component neutral component purified purified n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range direct interaction direct interaction 1E When co-purified, there are several peaks that can not be seen when the proteins are purified alone. ds_h4_rna-rep-3 1 5 enzyme target enzyme target substrate substrate neutral component neutral component 5' 32p radiolabel 32p radiolabel P32 32P 32P P32 certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) 6 stimulator stimulator neutral component neutral component purified purified n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) 7 enzyme enzyme neutral component neutral component purified purified P0DTD1-PRO_0000449631:p.Asp90Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain D A Thu Apr 22 10:28:16 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp90Ala' n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range P0DTD1-PRO_0000449631:p.Glu92Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain E A Thu Apr 22 10:38:59 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Glu92Ala' P0DTD1-PRO_0000449631:p.Asp243Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain D A Thu Apr 22 11:49:11 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp243Ala' P0DTD1-PRO_0000449631:p.Asp273Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain D A Thu Apr 22 12:46:01 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp273Ala' ecoli Escherichia coli (strain K12) rna cleavage rna cleavage 5C Co-purifying nsp14 and nsp10 proteins substantially increases nsp14 ExoNase activity. MnCl2 Mgcl2 Addition of ZnCl2 and CoCl2 caused a slight increase in ExoN activity. EDTA (CHEBI:42191 ) Mutations of nsp10 (with the exception of S72A) reduced ExoNase activity to that of nsp14 alone. The S72A mutation further decreased activity. rep-rep-149 3 7 enzyme enzyme neutral component neutral component purified purified n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range P0DTD1-PRO_0000449631:p.Asp90Ala 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 D A Thu Apr 22 12:39:56 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp90Ala' P0DTD1-PRO_0000449631:p.Glu92Ala 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 E A Thu Apr 22 10:40:23 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Glu92Ala' P0DTD1-PRO_0000449631:p.Asp243Ala 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 D A Thu Apr 22 12:28:07 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp243Ala' P0DTD1-PRO_0000449631:p.Asp273Ala 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 D A Thu Apr 22 13:29:24 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp273Ala' P0DTD1-PRO_0000449631:p.Asp331Ala mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain D A Thu Apr 22 11:45:37 BST 2021 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp331Ala' ecoli Escherichia coli (strain K12) 8 enzyme target enzyme target substrate substrate neutral component neutral component guanosine 32p radiolabel 32p radiolabel P32 32P 32P P32 certain certain sequence position certain certain certain sequence position certain in vitro In vitro methylation methylation reaction methylation 6 nsp14 MTase activity seems to be independent of the presence of nsp10 cofactor.