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/ Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2. Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2. The EMBO journal 2020 imex curation Klemm D et al., Klemm T., Ebert G., Calleja DJ., Allison CC., Richardson LW., Bernardini JP., Lu BG., Kuchel NW., Grohmann C., Shibata Y., Gan ZY., Cooney JP., Doerflinger M., Au AE., Blackmore TR., van der Heden van Noort GJ., Geurink PP., Ovaa H., Newman J., Riboldi-Tunnicliffe A., Czabotar PE., Mitchell JP., Feltham R., Lechtenberg BC., Lowes KN., Dewson G., Pellegrini M., Lessene G., Komander D. Coronavirus - Interactions investigated in the context of Coronavirus Virus - Publications including interactions involving viral proteins was unassigned2499 On publication the figure legends have changed a lot, and new interactions with UbiX2-lys48 construct. Figure 4D;self-cleavage of NSP3-GFP +/- inhibitor not entered, as I cannot see any purification from 293t cells. Email was sent to authors- No reply. (regarding K232E mutant and expression system + for the ISG15 Pro forms. 10.1101/2020.06.18.160614 dk@wehi.edu.au 28-Sep-2020: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Klemm D et al., Klemm T., Ebert G., Calleja DJ., Allison CC., Richardson LW., Bernardini JP., Lu BG., Kuchel NW., Grohmann C., Shibata Y., Gan ZY., Cooney JP., Doerflinger M., Au AE., Blackmore TR., van der Heden van Noort GJ., Geurink PP., Ovaa H., Newman J., Riboldi-Tunnicliffe A., Czabotar PE., Mitchell JP., Feltham R., Lechtenberg BC., Lowes KN., Dewson G., Pellegrini M., Lessene G., Komander D. Coronavirus - Interactions investigated in the context of Coronavirus was unassigned2499 On publication the figure legends have changed a lot, and new interactions with UbiX2-lys48 construct. dk@wehi.edu.au Accepted 2020-SEP-16 AT 13:03 BST AT 13:03 BST by MARTA The EMBO journal 2020 Only protein-protein interactions imex curation imex curation Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2. Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2. The EMBO journal 2020 imex curation Klemm D et al., Klemm T., Ebert G., Calleja DJ., Allison CC., Richardson LW., Bernardini JP., Lu BG., Kuchel NW., Grohmann C., Shibata Y., Gan ZY., Cooney JP., Doerflinger M., Au AE., Blackmore TR., van der Heden van Noort GJ., Geurink PP., Ovaa H., Newman J., Riboldi-Tunnicliffe A., Czabotar PE., Mitchell JP., Feltham R., Lechtenberg BC., Lowes KN., Dewson G., Pellegrini M., Lessene G., Komander D. Coronavirus - Interactions investigated in the context of Coronavirus Virus - Publications including interactions involving viral proteins was unassigned2499 On publication the figure legends have changed a lot, and new interactions with UbiX2-lys48 construct. Figure 4D;self-cleavage of NSP3-GFP +/- inhibitor not entered, as I cannot see any purification from 293t cells. Email was sent to authors- No reply. (regarding K232E mutant and expression system + for the ISG15 Pro forms. 10.1101/2020.06.18.160614 dk@wehi.edu.au 28-Sep-2020: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro cleavage cleavage assay cleavage predetermined predetermined participant predetermined Klemm D et al., Klemm T., Ebert G., Calleja DJ., Allison CC., Richardson LW., Bernardini JP., Lu BG., Kuchel NW., Grohmann C., Shibata Y., Gan ZY., Cooney JP., Doerflinger M., Au AE., Blackmore TR., van der Heden van Noort GJ., Geurink PP., Ovaa H., Newman J., Riboldi-Tunnicliffe A., Czabotar PE., Mitchell JP., Feltham R., Lechtenberg BC., Lowes KN., Dewson G., Pellegrini M., Lessene G., Komander D. Coronavirus - Interactions investigated in the context of Coronavirus it is unclear which TAMRA tagged construct is used - there is more than 1 Lys/Gly UB mols. was unassigned2499 On publication the figure legends have changed a lot, and new interactions with UbiX2-lys48 construct. Fluorescence polarisation (FP) assay; the hydrolysed product (Lys-TAMRA/(Tetramethylrhodamine)-Gly) emits much less polarized light than Ub-Lys-Gly. dk@wehi.edu.au Accepted 2020-SEP-16 AT 13:03 BST AT 13:03 BST by MARTA The EMBO journal 2020 Only protein-protein interactions imex curation imex curation Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2. Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2. The EMBO journal 2020 imex curation Klemm D et al., Klemm T., Ebert G., Calleja DJ., Allison CC., Richardson LW., Bernardini JP., Lu BG., Kuchel NW., Grohmann C., Shibata Y., Gan ZY., Cooney JP., Doerflinger M., Au AE., Blackmore TR., van der Heden van Noort GJ., Geurink PP., Ovaa H., Newman J., Riboldi-Tunnicliffe A., Czabotar PE., Mitchell JP., Feltham R., Lechtenberg BC., Lowes KN., Dewson G., Pellegrini M., Lessene G., Komander D. Coronavirus - Interactions investigated in the context of Coronavirus Virus - Publications including interactions involving viral proteins was unassigned2499 On publication the figure legends have changed a lot, and new interactions with UbiX2-lys48 construct. Figure 4D;self-cleavage of NSP3-GFP +/- inhibitor not entered, as I cannot see any purification from 293t cells. Email was sent to authors- No reply. (regarding K232E mutant and expression system + for the ISG15 Pro forms. 10.1101/2020.06.18.160614 dk@wehi.edu.au 28-Sep-2020: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro protease assay protease assay weight by comassie molecular weight estimation by coomasie staining weight by comassie Klemm D et al., Klemm T., Ebert G., Calleja DJ., Allison CC., Richardson LW., Bernardini JP., Lu BG., Kuchel NW., Grohmann C., Shibata Y., Gan ZY., Cooney JP., Doerflinger M., Au AE., Blackmore TR., van der Heden van Noort GJ., Geurink PP., Ovaa H., Newman J., Riboldi-Tunnicliffe A., Czabotar PE., Mitchell JP., Feltham R., Lechtenberg BC., Lowes KN., Dewson G., Pellegrini M., Lessene G., Komander D. Coronavirus - Interactions investigated in the context of Coronavirus No expression system is given for the ISG15 Pro forms; the pOPINB vector can be used in Insect, mammalian or bacterial systems. was unassigned2499 On publication the figure legends have changed a lot, and new interactions with UbiX2-lys48 construct. dk@wehi.edu.au Accepted 2020-SEP-16 AT 13:03 BST AT 13:03 BST by MARTA The EMBO journal 2020 Only protein-protein interactions imex curation imex curation Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2. Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2. The EMBO journal 2020 imex curation Klemm D et al., Klemm T., Ebert G., Calleja DJ., Allison CC., Richardson LW., Bernardini JP., Lu BG., Kuchel NW., Grohmann C., Shibata Y., Gan ZY., Cooney JP., Doerflinger M., Au AE., Blackmore TR., van der Heden van Noort GJ., Geurink PP., Ovaa H., Newman J., Riboldi-Tunnicliffe A., Czabotar PE., Mitchell JP., Feltham R., Lechtenberg BC., Lowes KN., Dewson G., Pellegrini M., Lessene G., Komander D. Coronavirus - Interactions investigated in the context of Coronavirus Virus - Publications including interactions involving viral proteins was unassigned2499 On publication the figure legends have changed a lot, and new interactions with UbiX2-lys48 construct. Figure 4D;self-cleavage of NSP3-GFP +/- inhibitor not entered, as I cannot see any purification from 293t cells. Email was sent to authors- No reply. (regarding K232E mutant and expression system + for the ISG15 Pro forms. 10.1101/2020.06.18.160614 dk@wehi.edu.au 28-Sep-2020: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro cleavage cleavage assay cleavage predetermined predetermined participant predetermined Klemm D et al., Klemm T., Ebert G., Calleja DJ., Allison CC., Richardson LW., Bernardini JP., Lu BG., Kuchel NW., Grohmann C., Shibata Y., Gan ZY., Cooney JP., Doerflinger M., Au AE., Blackmore TR., van der Heden van Noort GJ., Geurink PP., Ovaa H., Newman J., Riboldi-Tunnicliffe A., Czabotar PE., Mitchell JP., Feltham R., Lechtenberg BC., Lowes KN., Dewson G., Pellegrini M., Lessene G., Komander D. Coronavirus - Interactions investigated in the context of Coronavirus was unassigned2499 On publication the figure legends have changed a lot, and new interactions with UbiX2-lys48 construct. High Throughput Screening assay: Cleavage of the amide bond between the C-terminal glycine of ubiquitin and rhodamine110Gly (Rh110Gly) results in an increase in fluorescence. dk@wehi.edu.au Accepted 2020-SEP-16 AT 13:03 BST AT 13:03 BST by MARTA The EMBO journal 2020 Only protein-protein interactions imex curation imex curation rac5c rac5c small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) isg15_human Ubiquitin-like protein ISG15 ISG15 G1P2 UCRP Interferon-induced 15 kDa protein Interferon-induced 17 kDa protein Ubiquitin cross-reactive protein protein protein human Homo sapiens Human MGWDLTVKMLAGNEFQVSLSSSMSVSELKAQITQKIGVHAFQQRLAVHPSGVALQDRVPLASQGLGPGSTVLLVVDKCDEPLSILVRNNKGRSSTYEVRLTQTVAHLKQQVSGLEGVQDDLFWLTFEGKPLEDQLPLGEYGLKPLSTVFMNLRLRGGGTEPGGRS DIP protein P05161 original sequence version: 130 DIP protein P05161 original sequence version: 142 DIP protein P05161 original sequence version: 155 DIP protein P05161 original sequence version: 96 DIP protein P05161 original sequence version: 141 DIP protein P05161 original sequence version: 168 DIP protein P05161 original sequence version: 150 B6858A15AB0FFFDE j3qs39_human UBB protein protein human Homo sapiens Human MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEV 666B0F57D365A5A7 p0dtd1-pro_0000449621 Papain-like protease nsp3 rep ORF1ab polyprotein 1a-1b protein protein SARS-CoV-2 SARS-CoV-2 SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 2019-nCoV APTKVTFGDDTVIEVQGYKSVNITFELDERIDKVLNEKCSAYTVELGTEVNEFACVVADAVIKTLQPVSELLTPLGIDLDEWSMATYYLFDESGEFKLASHMYCSFYPPDEDEEEGDCEEEEFEPSTQYEYGTEDDYQGKPLEFGATSAALQPEEEQEEDWLDDDSQQTVGQQDGSEDNQTTTIQTIVEVQPQLEMELTPVVQTIEVNSFSGYLKLTDNVYIKNADIVEEAKKVKPTVVVNAANVYLKHGGGVAGALNKATNNAMQVESDDYIATNGPLKVGGSCVLSGHNLAKHCLHVVGPNVNKGEDIQLLKSAYENFNQHEVLLAPLLSAGIFGADPIHSLRVCVDTVRTNVYLAVFDKNLYDKLVSSFLEMKSEKQVEQKIAEIPKEEVKPFITESKPSVEQRKQDDKKIKACVEEVTTTLEETKFLTENLLLYIDINGNLHPDSATLVSDIDITFLKKDAPYIVGDVVQEGVLTAVVIPTKKAGGTTEMLAKALRKVPTDNYITTYPGQGLNGYTVEEAKTVLKKCKSAFYILPSIISNEKQEILGTVSWNLREMLAHAEETRKLMPVCVETKAIVSTIQRKYKGIKIQEGVVDYGARFYFYTSKTTVASLINTLNDLNETLVTMPLGYVTHGLNLEEAARYMRSLKVPATVSVSSPDAVTAYNGYLTSSSKTPEEHFIETISLAGSYKDWSYSGQSTQLGIEFLKRGDKSVYYTSNPTTFHLDGEVITFDNLKTLLSLREVRTIKVFTTVDNINLHTQVVDMSMTYGQQFGPTYLDGADVTKIKPHNSHEGKTFYVLPNDDTLRVEAFEYYHTTDPSFLGRYMSALNHTKKWKYPQVNGLTSIKWADNNCYLATALLTLQQIELKFNPPALQDAYYRARAGEAANFCALILAYCNKTVGELGDVRETMSYLFQHANLDSCKRVLNVVCKTCGQQQTTLKGVEAVMYMGTLSYEQFKKGVQIPCTCGKQATKYLVQQESPFVMMSAPPAQYELKHGTFTCASEYTGNYQCGHYKHITSKETLYCIDGALLTKSSEYKGPITDVFYKENSYTTTIKPVTYKLDGVVCTEIDPKLDNYYKKDNSYFTEQPIDLVPNQPYPNASFDNFKFVCDNIKFADDLNQLTGYKKPASRELKVTFFPDLNGDVVAIDYKHYTPSFKKGAKLLHKPIVWHVNNATNKATYKPNTWCIRCLWSTKPVETSNSFDVLKSEDAQGMDNLACEDLKPVSEEVVENPTIQKDVLECNVKTTEVVGDIILKPANNSLKITEEVGHTDLMAAYVDNSSLTIKKPNELSRVLGLKTLATHGLAAVNSVPWDTIANYAKPFLNKVVSTTTNIVTRCLNRVCTNYMPYFFTLLLQLCTFTRSTNSRIKASMPTTIAKNTVKSVGKFCLEASFNYLKSPNFSKLINIIIWFLLLSVCLGSLIYSTAALGVLMSNLGMPSYCTGYREGYLNSTNVTIATYCTGSIPCSVCLSGLDSLDTYPSLETIQITISSFKWDLTAFGLVAEWFLAYILFTRFFYVLGLAAIMQLFFSYFAVHFISNSWLMWLIINLVQMAPISAMVRMYIFFASFYYVWKSYVHVVDGCNSSTCMMCYKRNRATRVECTTIVNGVRRSFYVYANGGKGFCKLHNWNCVNCDTFCAGSTFISDEVARDLSLQFKRPINPTDQSSYIVDSVTVKNGSIHLYFDKAGQKTYERHSLSHFVNLDNLRANNTKGSLPINVIVFDGKSKCEESSAKSASVYYSQLMCQPILLLDQALVSDVGDSAEVAVKMFDAYVNTFSSTFNVPMEKLKTLVATAEAELAKNVSLDNVLSTFISAARQGFVDSDVETKDVVECLKLSHQSDIEVTGDSCNNYMLTYNKVENMTPRDLGACIDCSARHINAQVAKSHNIALIWNVKDFMSLSEQLRKQIRSAAKKNNLPFKLTCATTRQVVNVVTTKIALKGG 819 2763 isg15_mouse Ubiquitin-like protein ISG15 Isg15 G1p2 Ucrp Interferon-induced 15 kDa protein Interferon-induced 17 kDa protein Ubiquitin cross-reactive protein protein protein mouse Mus musculus Mouse MAWDLKVKMLGGNDFLVSVTNSMTVSELKKQIAQKIGVPAFQQRLAHQTAVLQDGLTLSSLGLGPSSTVMLVVQNCSEPLSILVRNERGHSNIYEVFLTQTVDTLKKKVSQREQVHEDQFWLSFEGRPMEDKELLGEYGLKPQCTVIKHLRLRGGGGDQCA In response to IFN-tau secreted by the conceptus, may ligate to and regulate proteins involved in the release of prostaglandin F2-alpha (PGF), and thus prevent lysis of the corpus luteum and maintain the pregnancy mint Ubiquitin-like protein that is conjugated to intracellular target proteins after IFN-alpha or IFN-beta stimulation. Its enzymatic pathway is partially distinct from that of ubiquitin, differing in substrate specificity and interaction with ligating enzymes. ISG15 conjugation pathway uses a dedicated E1 enzyme, but seems to converge with the Ub conjugation pathway at the level of a specific E2 enzyme. Targets include STAT1, SERPINA3G/SPI2A, JAK1, MAPK3/ERK1, PLCG1, EIF2AK2/PKR, MX1/MxA, and RIG-1. Deconjugated by USP18/UBP43. Shows specific chemotactic activity towards neutrophils and activates them to induce release of eosinophil chemotactic factors. May serve as a trans-acting binding factor directing the association of ligated target proteins to intermediate filaments. May also be involved in autocrine, paracrine and endocrine mechanisms, as in cell-to-cell signaling, possibly partly by inducing IFN-gamma secretion by monocytes and macrophages. Seems to display antiviral activity during viral infections 9C70D9A507979171 rep-94 1 7 unspecified role unspecified role neutral component neutral component purified purified propargylamide modification modgly modified glycine residue ModGly certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component purified purified PLPro_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction Table 1 isg15-rep-1 1 6 unspecified role unspecified role neutral component neutral component purified purified propargylamide modification modgly modified glycine residue ModGly certain certain sequence position certain certain certain sequence position certain ctd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component purified purified PLPro_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction Table 1 rep-ubb-1 2 7 enzyme target enzyme target substrate substrate neutral component neutral component purified purified lysine side chain tetramethyl rhodamine label tetramethyl rhodamine label undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chemical synthesis Chemical synthesis (Chemical synthesis) 8 enzyme enzyme neutral component neutral component purified purified P0DTD1-PRO_0000449621:p.Asn901Glu mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain N E Tue Jun 30 19:58:54 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asn901Glu' PLPro_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain P0DTD1-PRO_0000449621:p.Tyr916Arg mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain Y R Tue Jun 30 16:22:05 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Tyr916Arg' ecoli Escherichia coli (strain K12) cleavage cleavage reaction cleavage (1D),Appendix Figure S1A, 2B, Ext.Data 2A Kcat/Km =86/M/sec for WT;Y171R = 51/M/sec;N156E=62/M/sec PLpro N156E (and, to a lesser degree, Y171R) resulted in selective decrease of activity in ISG15 cleavage assays, with little impact on ubiquitin cleavage. isg15-rep-2 2 9 enzyme target enzyme target substrate substrate neutral component neutral component purified purified C-terminus tetramethyl rhodamine label tetramethyl rhodamine label c-term range C-terminal range c-term range c-term range C-terminal range c-term range region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain Q64339:p.[Cys76Ser;Cys144Ala] mutation mutation certain certain sequence position certain certain certain sequence position certain C S certain certain sequence position certain certain certain sequence position certain C A Tue Jun 30 18:29:16 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Cys76Ser;Cys144Ala]' synthetic construct synthetic construct 8 enzyme enzyme neutral component neutral component purified purified P0DTD1-PRO_0000449621:p.Asn901Glu mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain N E Tue Jun 30 16:45:51 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asn901Glu' P0DTD1-PRO_0000449621:p.Tyr916Arg mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain Y R Tue Jun 30 18:44:03 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Tyr916Arg' P0DTD1-PRO_0000449621:p.Arg911_Glu912delinsSerArg mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain RE SR Tue Jun 30 16:49:36 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg911_Glu912delinsSerArg' PLPro_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain P0DTD1-PRO_0000449621:p.Phe814Ser mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain F S Tue Jun 30 18:17:01 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe814Ser' ecoli Escherichia coli (strain K12) cleavage cleavage reaction cleavage (1D),Appendix Figure S1A, 2B, 3C, Ext.Data 2A, 6C Kcat/Km =30210 /M/sec WT; N156E=4803/M/sec;Y171R=8399 /M/sec;Arg166Ser/Glu167Arg =4336/M/sec;F69S=11690/M/sec. SARS2 PLpro hydrolysed an ISG15-TAMRA fluorescent substrate 350-fold more efficiently as compared to ubiquitin-TAMRA. PLpro N156E (and, to a lesser degree, Y171R) resulted in selective decrease of activity in ISG15 cleavage assays, with little impact on ubiquitin cleavage. isg15-rep-3 2 6 enzyme target enzyme target substrate substrate neutral component neutral component purified purified ctd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region tetramethyl rhodamine label tetramethyl rhodamine label undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 8 enzyme enzyme neutral component neutral component purified purified PLPro_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) cleavage cleavage reaction cleavage (1D),Appendix Figure S1A, Ext.Data 2A Kcat/Km =14040 /M/sec. A substrate comprising only the C-terminal ubiquitin-like fold of ISG15, (ISG15 CTD -TAMRA) was still cleaved with 160-fold higher efficiency compared to ubiquitin. rep-ubb-2 3 7 enzyme target enzyme target substrate substrate neutral component neutral component purified purified Lys48 cross linker cross linker crosslinker crosslinker certain certain sequence position certain certain certain sequence position certain true ecoli Escherichia coli (strain K12) 8 enzyme enzyme neutral component neutral component purified purified P0DTD1-PRO_0000449621:p.Phe814Ser mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain F S Tue Jun 30 17:08:24 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe814Ser' P0DTD1-PRO_0000449621:p.Arg911_Glu912delinsSerArg mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain RE SR Tue Jun 30 19:30:39 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg911_Glu912delinsSerArg' P0DTD1-PRO_0000449621:p.Asn901Glu 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 N E Tue Jun 30 15:59:38 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asn901Glu' P0DTD1-PRO_0000449621:p.Tyr916Arg 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 Y R Tue Jun 30 16:46:18 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Tyr916Arg' P0DTD1-PRO_0000449621:p.Lys977Glu mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain K E Tue Jun 30 20:11:43 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys977Glu' P0DTD1-PRO_0000449621:p.Thr970Tyr mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain T Y Tue Jun 30 18:13:34 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Thr970Tyr' PLPro_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) protein cleavage protein cleavage Appendix Fig S1B, 2C, Figure 3D, 5 E/F, 6E Very high activity and striking Lys48-polyubiquitin specificity was observed for SARS2 PLpro. rep-ubb-3 3 6 enzyme target enzyme target substrate substrate neutral component neutral component purified purified P05161:p.Cys78Ser mutation mutation certain certain sequence position certain certain certain sequence position certain C S Tue Jun 30 17:49:18 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys78Ser' Pro_form sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 8 enzyme enzyme neutral component neutral component purified purified P0DTD1-PRO_0000449621:p.Asn901Glu mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain N E Tue Jun 30 16:22:06 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asn156Glu' PLPro_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain P0DTD1-PRO_0000449621:p.Tyr916Arg mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain Y R Tue Jun 30 16:43:36 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Tyr916Arg' P0DTD1-PRO_0000449621:p.Arg911_Glu912delinsSerArg mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain RE SR Tue Jun 30 17:55:26 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg911_Glu912delinsSerArg' P0DTD1-PRO_0000449621:p.Lys977Glu 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 K E Tue Jun 30 17:22:52 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys977Glu' P0DTD1-PRO_0000449621:p.Thr970Tyr 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 T Y Tue Jun 30 19:47:47 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Thr970Tyr' P0DTD1-PRO_0000449621:p.Phe814Ser mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain F S Tue Jun 30 19:44:36 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe814Ser' ecoli Escherichia coli (strain K12) protein cleavage protein cleavage Appendix Fig S1C, EV3-E/F,Figure 3F, Ext. Data 2C,5 E/F The K232E (p.Lys977Glu) mutation (Ext. fig 5); I am not sure if I am going nuts here looking at proISG15 gel - the text states that:-"Mutations selectively impacting ubiquitin but not ISG15 were more challenging to generate but were apparent by gel-based analysis (see e.g. K232E)". I would read this as meaning " a mutation of NO effect" for proISG15, but from looking at the gel, I would read this as either a mutation "increasing" (at 0.5 mins all the bands has gone to the lower M.Wt -but this might be an artefact), OR it is a mutation "decreasing" as the no proteolysis is occurring at all - all bands are the same M. Wt. (maybe we need a "mutation of unknown effect" to add to the list!) Maybe the mutation should be removed? I have added a comment regarding this mutation - please do what you think fit.. The K232E (p.Lys977Glu) mutation (Ext. fig 5); it is unclear what the effect of this mutation is on proISG15 cleavage. isg15-rep-5 3 6 enzyme target enzyme target substrate substrate neutral component neutral component purified purified P05161:p.Cys78Ser mutation mutation certain certain sequence position certain certain certain sequence position certain C S Tue Jun 30 17:49:26 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys78Ser' CTD_Pro_form sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 8 enzyme enzyme neutral component neutral component purified purified P0DTD1-PRO_0000449621:p.Phe814Ser mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain F S Tue Jun 30 17:21:52 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe814Ser' PLPro_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) protein cleavage protein cleavage Appendix Fig S1D, Figure 3E, Ext. Data 2D,6D cleavage was less rapid with the Pro-CTD construct than the 2-165 construct. rep-ubb-4 4 7 enzyme target enzyme target substrate substrate neutral component neutral component purified purified C-terminus tetramethyl rhodamine label tetramethyl rhodamine label c-term range C-terminal range c-term range c-term range C-terminal range c-term range ecoli Escherichia coli (strain K12) 8 enzyme enzyme neutral component neutral component purified purified PLPro_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) cleavage cleavage reaction cleavage 4B, EV4 rep-ubb-5 4 5 inhibitor inhibitor neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 7 enzyme target enzyme target substrate substrate neutral component neutral component purified purified C-terminus tetramethyl rhodamine label tetramethyl rhodamine label c-term range C-terminal range c-term range c-term range C-terminal range c-term range chemical synthesis Chemical synthesis (Chemical synthesis) 8 enzyme enzyme neutral component neutral component purified purified PLPro_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) cleavage cleavage reaction cleavage 4B, 5C rep-ubb-6 2 7 enzyme target enzyme target substrate substrate neutral component neutral component purified purified lysine-48 tetramethyl rhodamine label tetramethyl rhodamine label undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chemical synthesis Chemical synthesis (Chemical synthesis) 8 enzyme enzyme neutral component neutral component purified purified P0DTD1-PRO_0000449621:p.Asn901Glu mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain N E Tue Jun 30 19:58:54 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asn901Glu' PLPro_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain P0DTD1-PRO_0000449621:p.Tyr916Arg mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain Y R Tue Jun 30 16:22:05 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Tyr916Arg' P0DTD1-PRO_0000449621:p.Phe814Ser mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain F S ecoli Escherichia coli (strain K12) cleavage cleavage reaction cleavage (1D),Appendix Figure S1A, figure 3C KG‐TAMRA peptide was hydrolysed 19‐fold faster - Kcat/Km=1,634/M/sec, when linked to a non‐cleavable Lys48‐diubiquitin. PLpro F69S Kcat/Km=45/M/Sec