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/ Specific recognition of a multiply phosphorylated motif in the DNA repair scaffold XRCC1 by the FHA domain of human PNK. Specific recognition of a multiply phosphorylated motif in the DNA repair scaffold XRCC1 by the FHA domain of human PNK. Nucleic Acids Res. (0305-1048) 2009 imex curation Ali A.A., Jukes R.M., Pearl L.H., Oliver A.W. antony.oliver@icr.ac.uk,laurence.pearl@icr.ac.uk 28-Oct_2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Ali A.A., Jukes R.M., Pearl L.H., Oliver A.W. antony.oliver@icr.ac.uk,laurence.pearl@icr.ac.uk Accepted 2013-OCT-21 by ORCHARD 28-Oct_2013: Contacted by IntAct-Help. Nucleic Acids Res. (0305-1048) 2009 Only protein-protein interactions imex curation Specific recognition of a multiply phosphorylated motif in the DNA repair scaffold XRCC1 by the FHA domain of human PNK. Specific recognition of a multiply phosphorylated motif in the DNA repair scaffold XRCC1 by the FHA domain of human PNK. Nucleic Acids Res. (0305-1048) 2009 imex curation Ali A.A., Jukes R.M., Pearl L.H., Oliver A.W. antony.oliver@icr.ac.uk,laurence.pearl@icr.ac.uk 28-Oct_2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Ali A.A., Jukes R.M., Pearl L.H., Oliver A.W. antony.oliver@icr.ac.uk,laurence.pearl@icr.ac.uk Accepted 2013-OCT-21 by ORCHARD 28-Oct_2013: Contacted by IntAct-Help. Nucleic Acids Res. (0305-1048) 2009 Only protein-protein interactions imex curation Specific recognition of a multiply phosphorylated motif in the DNA repair scaffold XRCC1 by the FHA domain of human PNK. Specific recognition of a multiply phosphorylated motif in the DNA repair scaffold XRCC1 by the FHA domain of human PNK. Nucleic Acids Res. (0305-1048) 2009 imex curation Ali A.A., Jukes R.M., Pearl L.H., Oliver A.W. antony.oliver@icr.ac.uk,laurence.pearl@icr.ac.uk 28-Oct_2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro itc isothermal titration calorimetry ITC ITC itc predetermined predetermined participant predetermined Ali A.A., Jukes R.M., Pearl L.H., Oliver A.W. antony.oliver@icr.ac.uk,laurence.pearl@icr.ac.uk Accepted 2013-OCT-21 by ORCHARD 28-Oct_2013: Contacted by IntAct-Help. Nucleic Acids Res. (0305-1048) 2009 Only protein-protein interactions imex curation Specific recognition of a multiply phosphorylated motif in the DNA repair scaffold XRCC1 by the FHA domain of human PNK. Specific recognition of a multiply phosphorylated motif in the DNA repair scaffold XRCC1 by the FHA domain of human PNK. Nucleic Acids Res. (0305-1048) 2009 imex curation Ali A.A., Jukes R.M., Pearl L.H., Oliver A.W. antony.oliver@icr.ac.uk,laurence.pearl@icr.ac.uk 28-Oct_2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay western blot western blot Immuno blot Immuno blot Ali A.A., Jukes R.M., Pearl L.H., Oliver A.W. antony.oliver@icr.ac.uk,laurence.pearl@icr.ac.uk Accepted 2013-OCT-21 by ORCHARD 28-Oct_2013: Contacted by IntAct-Help. Nucleic Acids Res. (0305-1048) 2009 Only protein-protein interactions imex curation pnkp_human Bifunctional polynucleotide phosphatase/kinase Polynucleotide kinase-3'-phosphatase DNA 5'-kinase/3'-phosphatase PNKP protein protein human Homo sapiens Human MGEVEAPGRLWLESPPGGAPPIFLPSDGQALVLGRGPLTQVTDRKCSRTQVELVADPETRTVAVKQLGVNPSTTGTQELKPGLEGSLGVGDTLYLVNGLHPLTLRWEETRTPESQPDTPPGTPLVSQDEKRDAELPKKRMRKSNPGWENLEKLLVFTAAGVKPQGKVAGFDLDGTLITTRSGKVFPTGPSDWRILYPEIPRKLRELEAEGYKLVIFTNQMSIGRGKLPAEEFKAKVEAVVEKLGVPFQVLVATHAGLYRKPVTGMWDHLQEQANDGTPISIGDSIFVGDAAGRPANWAPGRKKKDFSCADRLFALNLGLPFATPEEFFLKWPAAGFELPAFDPRTVSRSGPLCLPESRALLSASPEVVVAVGFPGAGKSTFLKKHLVSAGYVHVNRDTLGSWQRCVTTCETALKQGKRVAIDNTNPDAASRARYVQCARAAGVPCRCFLFTATLEQARHNNRFREMTDSSHIPVSDMVMYGYRKQFEAPTLAEGFSAILEIPFRLWVEPRLGRLYCQFSEG 22B5C94D41E62516 csk2a_maize Casein kinase II subunit alpha ACK2 CK II CK2-alpha protein protein maize Zea mays (Maize) Maize MSKARVYADVNVLRPKEYWDYEALTVQWGEQDDYEVVRKVGRGKYSEVFEGINVNNNEKCIIKILKPVKKKKIKREIKILQNLCGGPNIVKLLDIVRDQHSKTPSLIFEYVNNTDFKVLYPTLTDYDIRYYIYELLKALDYCHSQGIMHRDVKPHNVMIDHELRKLRLIDWGLAEFYHPGKEYNVRVASRYFKGPELLVDLQDYDYSLDMWSLGCMFAGMIFRKEPFFYGHDNHDQLVKIAKVLGTDGLNVYLNKYRIELDPQLEALVGRHSRKPWLKFMNADNQHLVSPEAIDFLDKLLRYDHQERLTALEAMTHPYFQQVRAAENSRTRA 85513A5A5C77235A xrcc1_human DNA repair protein XRCC1 XRCC1 X-ray repair cross-complementing protein 1 protein protein human Homo sapiens Human MPEIRLRHVVSCSSQDSTHCAENLLKADTYRKWRAAKAGEKTISVVLQLEKEEQIHSVDIGNDGSAFVEVLVGSSAGGAGEQDYEVLLVTSSFMSPSESRSGSNPNRVRMFGPDKLVRAAAEKRWDRVKIVCSQPYSKDSPFGLSFVRFHSPPDKDEAEAPSQKVTVTKLGQFRVKEEDESANSLRPGALFFSRINKTSPVTASDPAGPSYAAATLQASSAASSASPVSRAIGSTSKPQESPKGKRKLDLNQEEKKTPSKPPAQLSPSVPKRPKLPAPTRTPATAPVPARAQGAVTGKPRGEGTEPRRPRAGPEELGKILQGVVVVLSGFQNPFRSELRDKALELGAKYRPDWTRDSTHLICAFANTPKYSQVLGLGGRIVRKEWVLDCHRMRRRLPSRRYLMAGPGSSSEEDEASHSGGSGDEAPKLPQKQPQTKTKPTQAAGPSSPQKPPTPEETKAASPVLQEDIDIEGVQSEGQDNGAEDSGDTEDELRRVAEQKEHRLPPGQEENGEDPYAGSTDENTDSEEHQEPPDLPVPELPDFFQGKHFFLYGEFPGDERRKLIRYVTAFNGELEDNMSDRVQFVITAQEWDPSFEEALMDNPSLAFVRPRWIYSCNEKQKLLPHQLYGVVPQA DIP protein P18887 original sequence version: 120 DIP protein P18887 original sequence version: 161 30DB3321234DBFC2 pnkp-xrcc1-1 2 5 unspecified role unspecified role neutral component neutral component fha domain sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain Q96T60:p.Leu99Glu mutation mutation certain certain sequence position certain certain certain sequence position certain L E Wed May 02 15:21:37 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'leu99glu' Tue Jun 30 19:37:42 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Leu99Glu' The leu99glu mutation disrupts a hydrophobic crystal packing interface, which is distant from the peptide-binding site but resulted in formation of a crystal form incompatible with retention of the peptide ligand. By using this mutant, formation of this crystal form was prevented and crystals of the peptide-containing complex could be obtained. 562-rosetta ii de3 Escherichia coli rosetta ii de3 rosetta ii de3 7 unspecified role unspecified role neutral component neutral component possible phosphoser-518 opser O-phospho-L-serine (2S)-2-amino-3-(phosphonooxy)propanoic acid 2-amino-3-hydroxypropanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)propanoic acid ACT_SITE Phosphoserine intermediate MOD_RES Phosphoserine O-phospho-L-serine O-phosphonoserine O-phosphorylated L-serine O3-phosphoserine serine phosphate ester OPSer Phospho Phospho Seryl Phosphorylation phosphorylations certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain required phosphothr-519 opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt certain certain sequence position certain certain certain sequence position certain possible phosphothr-523 opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) 10 14 direct interaction direct interaction Fig. 3, Fig. 4a, Fig. 5a, Suppl. Table S1 working 18%, free 24% 1.85A Residues 523 to 526 of the XRCC1 peptide are not visible in the structure. pnkp-xrcc1-2 3 5 unspecified role unspecified role bait bait fha domain sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 7 unspecified role unspecified role prey prey possible phosphothr-523 opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain required phosphothr-519 opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt certain certain sequence position certain certain certain sequence position certain possible phosphoser-518 opser O-phospho-L-serine (2S)-2-amino-3-(phosphonooxy)propanoic acid 2-amino-3-hydroxypropanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)propanoic acid ACT_SITE Phosphoserine intermediate MOD_RES Phosphoserine O-phospho-L-serine O-phosphonoserine O-phosphorylated L-serine O3-phosphoserine serine phosphate ester OPSer Phospho Phospho Seryl Phosphorylation phosphorylations certain certain sequence position certain certain certain sequence position certain possible phosphoser-525 opser O-phospho-L-serine (2S)-2-amino-3-(phosphonooxy)propanoic acid 2-amino-3-hydroxypropanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)propanoic acid ACT_SITE Phosphoserine intermediate MOD_RES Phosphoserine O-phospho-L-serine O-phosphonoserine O-phosphorylated L-serine O3-phosphoserine serine phosphate ester OPSer Phospho Phospho Seryl Phosphorylation phosphorylations certain certain sequence position certain certain certain sequence position certain P18887:p.Thr523Val mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain T V Wed May 02 17:45:09 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'thr523val' Tue Jun 30 16:27:10 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Thr523Val' chemical synthesis Chemical synthesis (Chemical synthesis) 19 22 direct interaction direct interaction Fig. 2a, Fig. 2b, Fig. 2c, Fig. 2e, Table 1 Kd values were determined for several XRCC1 phosphopeptides (515-526). No phosphorylated residues: no binding; pS518: no binding; pS525: no binding; pT523: Kd = 13.49 +/- 1.296 x 10e-6 M; pT519-pT523: Kd = 0.76 +/- 0.22 x 10e-6 M; pS518-pT519: Kd = 1.23 +/- 0.04 x 10e-6 M; pS518-pT519-pT523: Kd = 0.19 +/- 0.02 x 10e-6 M; pS518-pT519-pT523-pS525: Kd = 0.14 +/- 0.01 x 10e-6 M; pS518-pT523: Kd = 24.18 +/- 1.44 x 10e-6 M. The thr523val mutation in the pS518-pT519 peptide lowered the affinity to Kd = 1.7 +/- 0.06 x 10e-6 M. In contrast to all other peptides, the pT523 and pS518-pT519-thr523val peptides bound with a 1:1 stoichiometry. xrcc1-pnkp-1 3 5 unspecified role unspecified role prey prey fha domain sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 7 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain required phosphothr-519 opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt certain certain sequence position certain certain certain sequence position certain possible phosphoser-518 opser O-phospho-L-serine (2S)-2-amino-3-(phosphonooxy)propanoic acid 2-amino-3-hydroxypropanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)propanoic acid ACT_SITE Phosphoserine intermediate MOD_RES Phosphoserine O-phospho-L-serine O-phosphonoserine O-phosphorylated L-serine O3-phosphoserine serine phosphate ester OPSer Phospho Phospho Seryl Phosphorylation phosphorylations certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) 29 31 direct interaction direct interaction Fig. 2d pnkp-xrcc1-3 1 5 unspecified role unspecified role bait bait n-terminal gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range fha domain sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 562-rosetta ii de3 Escherichia coli rosetta ii de3 rosetta ii de3 7 unspecified role unspecified role prey prey n-terminal 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 region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection 40 37 direct interaction direct interaction Fig. 1d ack2-xrcc1 4 6 enzyme enzyme unspecified role unspecified role 562-rosetta ii de3 Escherichia coli rosetta ii de3 rosetta ii de3 7 enzyme target enzyme target substrate substrate unspecified role unspecified role region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 562-rosetta ii de3 Escherichia coli rosetta ii de3 rosetta ii de3 phosphorylation phosphorylation reaction phosphorylation Fig. 1c phosphorylation of XRCC1 on ser518, thr519 and thr523