IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ http://www.ebi.ac.uk/ de-2010-1 Molecular basis of the death-associated protein kinase-calcium/calmodulin regulator complex. in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined de Diego I., Kuper J., Bakalova N., Kursula P., Wilmanns M. IBD - Inflammatory bowel disease wilmanns@embl-hamburg.de Sci Signal 2010 Only protein-protein interactions imex curation Ulcerative colitis - Interactions of proteins identified as having a link to ulcerative colitis de-2010-4 Molecular basis of the death-associated protein kinase-calcium/calmodulin regulator complex. in vitro In vitro protein kinase assay protein kinase assay predetermined predetermined participant predetermined de Diego I., Kuper J., Bakalova N., Kursula P., Wilmanns M. IBD - Inflammatory bowel disease The synthetic DAPK consensus substrate peptide is described in PMID:11483604. The protein kinase assay is described in more detail in PMID:6295440. wilmanns@embl-hamburg.de Sci Signal 2010 Only protein-protein interactions imex curation Ulcerative colitis - Interactions of proteins identified as having a link to ulcerative colitis de-2010-5 Molecular basis of the death-associated protein kinase-calcium/calmodulin regulator complex. in vitro In vitro protein kinase assay protein kinase assay western blot western blot Immuno blot Immuno blot de Diego I., Kuper J., Bakalova N., Kursula P., Wilmanns M. IBD - Inflammatory bowel disease wilmanns@embl-hamburg.de Sci Signal 2010 Only protein-protein interactions imex curation Ulcerative colitis - Interactions of proteins identified as having a link to ulcerative colitis de-2010-2 Molecular basis of the death-associated protein kinase-calcium/calmodulin regulator complex. in vitro In vitro itc isothermal titration calorimetry ITC ITC itc predetermined predetermined participant predetermined de Diego I., Kuper J., Bakalova N., Kursula P., Wilmanns M. IBD - Inflammatory bowel disease wilmanns@embl-hamburg.de Sci Signal 2010 Only protein-protein interactions imex curation Ulcerative colitis - Interactions of proteins identified as having a link to ulcerative colitis de-2010-3 Molecular basis of the death-associated protein kinase-calcium/calmodulin regulator complex. ecobd Escherichia coli (strain B / BL21-DE3) molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column weight by staining molecular weight estimation by staining weight by staining de Diego I., Kuper J., Bakalova N., Kursula P., Wilmanns M. IBD - Inflammatory bowel disease wilmanns@embl-hamburg.de Sci Signal 2010 Only protein-protein interactions imex curation Ulcerative colitis - Interactions of proteins identified as having a link to ulcerative colitis calm_human Calmodulin CALM1 CALM CAM CAM1 CALM2 CAMB CALM3 CAM3 CAMC CAMIII CAM2 CALML2 protein protein human Homo sapiens Human MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK dapk1_human Death-associated protein kinase 1 DAPK1 DAPK protein protein human Homo sapiens Human MTVFRQENVDDYYDTGEELGSGQFAVVKKCREKSTGLQYAAKFIKKRRTKSSRRGVSREDIEREVSILKEIQHPNVITLHEVYENKTDVILILELVAGGELFDFLAEKESLTEEEATEFLKQILNGVYYLHSLQIAHFDLKPENIMLLDRNVPKPRIKIIDFGLAHKIDFGNEFKNIFGTPEFVAPEIVNYEPLGLEADMWSIGVITYILLSGASPFLGDTKQETLANVSAVNYEFEDEYFSNTSALAKDFIRRLLVKDPKKRMTIQDSLQHPWIKPKDTQQALSRKASAVNMEKFKKFAARKKWKQSVRLISLCQRLSRSFLSRSNMSVARSDDTLDEEDSFVMKAIIHAINDDNVPGLQHLLGSLSNYDVNQPNKHGTPPLLIAAGCGNIQILQLLIKRGSRIDVQDKGGSNAVYWAARHGHVDTLKFLSENKCPLDVKDKSGEMALHVAARYGHADVAQLLCSFGSNPNIQDKEEETPLHCAAWHGYYSVAKALCEAGCNVNIKNREGETPLLTASARGYHDIVECLAEHGADLNACDKDGHIALHLAVRRCQMEVIKTLLSQGCFVDYQDRHGNTPLHVACKDGNMPIVVALCEANCNLDISNKYGRTPLHLAANNGILDVVRYLCLMGASVEALTTDGKTAEDLARSEQHEHVAGLLARLRKDTHRGLFIQQLRPTQNLQPRIKLKLFGHSGSGKTTLVESLKCGLLRSFFRRRRPRLSSTNSSRFPPSPLASKPTVSVSINNLYPGCENVSVRSRSMMFEPGLTKGMLEVFVAPTHHPHCSADDQSTKAIDIQNAYLNGVGDFSVWEFSGNPVYFCCYDYFAANDPTSIHVVVFSLEEPYEIQLNQVIFWLSFLKSLVPVEEPIAFGGKLKNPLQVVLVATHADIMNVPRPAGGEFGYDKDTSLLKEIRNRFGNDLHISNKLFVLDAGASGSKDMKVLRNHLQEIRSQIVSVCPPMTHLCEKIISTLPSWRKLNGPNQLMSLQQFVYDVQDQLNPLASEEDLRRIAQQLHSTGEINIMQSETVQDVLLLDPRWLCTNVLGKLLSVETPRALHHYRGRYTVEDIQRLVPDSDVEELLQILDAMDICARDLSSGTMVDVPALIKTDNLHRSWADEEDEVMVYGGVRIVPVEHLTPFPCGIFHKVQVNLCRWIHQQSTEGDADIRLWVNGCKLANRGAELLVLLVNHGQGIEVQVRGLETEKIKCCLLLDSVCSTIENVMATTLPGLLTVKHYLSPQQLREHHEPVMIYQPRDFFRAQTLKETSLTNTMGGYKESFSSIMCFGCHDVYSQASLGMDIHASDLNLLTRRKLSRLLDPPDPLGKDWCLLAMNLGLPDLVAKYNTSNGAPKDFLPSPLHALLREWTTYPESTVGTLMSKLRELGRRDAADFLLKASSVFKINLDGNGQEAYASSCNSGTSYNSISSVVSR dap_kinase_substrate_peptide Death-associated protein kinase (DAPK) consensus substrate peptide peptide peptide polypeptide oligopeptide oligopeptide polypeptide chemical synthesis Chemical synthesis (Chemical synthesis) KKRPQRRYSNVF calm1-dapk1 1571197 n/a 1571202 unspecified role unspecified role neutral component neutral component ef-hand-dom_pair sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecobb Escherichia coli (strain B / BL21) Stoichiometry: 1.0 n/a 1571203 unspecified role unspecified role neutral component neutral component ser/thr_dual-sp_kinase sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecobb Escherichia coli (strain B / BL21) Stoichiometry: 1.0 direct interaction direct interaction false false false Table 1 2.20A working 20.1%, free 26.7% P62158 increases activity of P53355 calm1-dapk1-1 1571197 n/a 1571203 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) Stoichiometry: 1.0 n/a 1571202 unspecified role unspecified role neutral component neutral component ef-hand-dom_pair sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecobd Escherichia coli (strain B / BL21-DE3) Stoichiometry: 1.0 direct interaction direct interaction false false false Table 1 1.70A working 20.3%, free 25.7% dap-dapk1 1571198 n/a 1571204 enzyme target enzyme target substrate substrate neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) n/a 1571203 enzyme enzyme neutral component neutral component ser/thr_dual-sp_kinase sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecobd Escherichia coli (strain B / BL21-DE3) phosphorylation phosphorylation reaction phosphorylation false false false 1571198 1571198 Fig. 5b, Table 3 Temp = 30C, pH = 7.2 Peptide substrate was phosphorylated by constitutively active DAPK (residues 1-277), while the apo version of DAPK (residues 1-334) showed little catalytic activity (kcat = 2.0 +/- 0.2 x 10E-1 s-1). calm1-dap-4 1571198 n/a 1571204 enzyme target enzyme target substrate substrate neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) n/a 1571202 stimulator stimulator neutral component neutral component ecobd Escherichia coli (strain B / BL21-DE3) n/a 1571203 enzyme enzyme neutral component neutral component P53355:p.Trp305Tyr mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false P53355:p.Trp305Asp mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false ser/thr_dual-sp_kinase sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecobd Escherichia coli (strain B / BL21-DE3) phosphorylation phosphorylation reaction phosphorylation false false false Fig. 5b, Table 3 Temp = 30C, pH = 7.2 Mutations of the trp305 residue in DAPK decreased the catalytic activity of DAPK towards the peptide substrate: for the trp305tyr mutant kcat = 9.8 +/- 0.3 x 10E-1 s-1; for the trp305asp mutant kcat = 2.6 +/- 0.1 x 10E-1 s-1. calm1-dapk1-4 1571199 n/a 1571203 putative self putative self putative self putative self ser/thr_dual-sp_kinase sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P53355:p.Trp305Asp mutation increasing rate mutation increasing interaction rate mutation increasing rate mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false P53355:p.Ser308Ala mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false ecobd Escherichia coli (strain B / BL21-DE3) n/a 1571202 inhibitor inhibitor neutral component neutral component ecobd Escherichia coli (strain B / BL21-DE3) phosphorylation phosphorylation reaction phosphorylation false false false Fig. 5c P53355 ser-308 O-phospho-L-serine dapk1-4 1571199 n/a 1571203 putative self putative self putative self putative self ser/thr_dual-sp_kinase sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P53355:p.Ser308Ala mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false ecobd Escherichia coli (strain B / BL21-DE3) phosphorylation phosphorylation reaction phosphorylation false true false Fig. 5c P53355 ser-308 O-phospho-L-serine calm1-dapk1-2 1571200 n/a 1571202 unspecified role unspecified role neutral component neutral component ecobd Escherichia coli (strain B / BL21-DE3) Stoichiometry: 1.0 n/a 1571203 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) Stoichiometry: 1.0 direct interaction direct interaction false false false 1571200 Fig. S3, Table S1 Temp = 25C, pH = 7.4 In this experiment, mutation of the trp305 residue in DAPK has little effect on CaM binding: the trp305tyr mutant binds with Kd = 1.1 +/- 0.2 x 10E-8 molar, while the trp305asp mutant binds with Kd = 9.9 +/- 0.9 x 10E-9 molar. calm1-dapk1-3 1571201 n/a 1571202 unspecified role unspecified role neutral component neutral component n/a 1571203 unspecified role unspecified role neutral component neutral component P53355:p.Trp305Asp mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false P53355:p.Trp305Tyr mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false ser/thr_dual-sp_kinase sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Fig. 5a, 5d