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/ ziechner-2006-2 Inhibition of human ether a go-go potassium channels by Ca2+/calmodulin binding to the cytosolic N- and C-termini. in vitro In vitro fcs fluorescence correlation spectroscopy FCS FCS fcs tag fluorescence tag visualisation by fluorescence tag fluorescence Ziechner U., Schönherr R., Born A.K., Gavrilova-Ruch O., Glaser R.W., Malesevic M., Küllertz G., Heinemann S.H. IBD - Inflammatory bowel disease Cloning of full-length cDNA encoding hCaM (P62158) and hEAG1 (O95259-2) is described in PMID:10880439. (Ref 6) stefan.h.heinemann@uni-jena.de FEBS J. (1742-464X) 2006 Only protein-protein interactions imex curation Ulcerative colitis - Interactions of proteins identified as having a link to ulcerative colitis ziechner-2006-1 Inhibition of human ether a go-go potassium channels by Ca2+/calmodulin binding to the cytosolic N- and C-termini. in vitro In vitro pull down pull down weight by comassie molecular weight estimation by coomasie staining weight by comassie Ziechner U., Schönherr R., Born A.K., Gavrilova-Ruch O., Glaser R.W., Malesevic M., Küllertz G., Heinemann S.H. IBD - Inflammatory bowel disease Cloning of full-length cDNA encoding hCaM (P62158) and hEAG1 (O95259-2) is described in PMID:10880439. (Ref 6) stefan.h.heinemann@uni-jena.de FEBS J. (1742-464X) 2006 Only protein-protein interactions imex curation Ulcerative colitis - Interactions of proteins identified as having a link to ulcerative colitis ziechner-2006-3 Inhibition of human ether a go-go potassium channels by Ca2+/calmodulin binding to the cytosolic N- and C-termini. in vitro In vitro peptide array peptide array tag fluorescence tag visualisation by fluorescence tag fluorescence Ziechner U., Schönherr R., Born A.K., Gavrilova-Ruch O., Glaser R.W., Malesevic M., Küllertz G., Heinemann S.H. IBD - Inflammatory bowel disease stefan.h.heinemann@uni-jena.de FEBS J. (1742-464X) 2006 Only protein-protein interactions imex curation Ulcerative colitis - Interactions of proteins identified as having a link to ulcerative colitis ziechner-2006-4 Inhibition of human ether a go-go potassium channels by Ca2+/calmodulin binding to the cytosolic N- and C-termini. in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Ziechner U., Schönherr R., Born A.K., Gavrilova-Ruch O., Glaser R.W., Malesevic M., Küllertz G., Heinemann S.H. IBD - Inflammatory bowel disease stefan.h.heinemann@uni-jena.de FEBS J. (1742-464X) 2006 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 o95259-2 Potassium voltage-gated channel subfamily H member 1 hEAG KCNH1 EAG EAG1 Ether-a-go-go potassium channel 1 Voltage-gated potassium channel subunit Kv10.1 protein protein human Homo sapiens Human MTMAGGRRGLVAPQNTFLENIVRRSNDTNFVLGNAQIVDWPIVYSNDGFCKLSGYHRAEVMQKSSTCSFMYGELTDKDTIEKVRQTFENYEMNSFEILMYKKNRTPVWFFVKIAPIRNEQDKVVLFLCTFSDITAFKQPIEDDSCKGWGKFARLTRALTSSRGVLQQLAPSVQKGENVHKHSRLAEVLQLGSDILPQYKQEAPKTPPHIILHYCVFKTTWDWIILILTFYTAILVPYNVSFKTRQNNVAWLVVDSIVDVIFLVDIVLNFHTTFVGPAGEVISDPKLIRMNYLKTWFVIDLLSCLPYDVINAFENVDEGISSLFSSLKVVRLLRLGRVARKLDHYIEYGAAVLVLLVCVFGLAAHWMACIWYSIGDYEIFDEDTKTIRNNSWLYQLAMDIGTPYQFNGSGSGKWEGGPSKNSVYISSLYFTMTSLTSVGFGNIAPSTDIEKIFAVAIMMIGSLLYATIFGNVTTIFQQMYANTNRYHEMLNSVRDFLKLYQVPKGLSERVMDYIVSTWSMSRGIDTEKVLQICPKDMRADICVHLNRKVFKEHPAFRLASDGCLRALAMEFQTVHCAPGDLIYHAGESVDSLCFVVSGSLEVIQDDEVVAILGKGDVFGDVFWKEATLAQSCANVRALTYCDLHVIKRDALQKVLEFYTAFSHSFSRNLILTYNLRKRIVFRKISDVKREEEERMKRKNEAPLILPPDHPVRRLFQRFRQQKEARLAAERGGRDLDDLDVEKGNVLTEHASANHSLVKASVVTVRESPATPVSFQAASTSGVPDHAKLQAPGSECLGPKGGGGDCAKRKSWARFKDACGKSEDWNKVSKAESMETLPERTKASGEATLKKTDSCDSGITKSDLRLDNVGEARSPQDRSPILAEVKHSFYPIPEQTLQATVLEVRHELKEDIKALNAKMTNIEKQLSEILRILTSRRSSQSPQELFEISRPQSPESERDIFGAS calm1-kcnh1-2 1571685 n/a 1571689 unspecified role unspecified role neutral component neutral component 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 false n-terminus tetramethyl rhodamine label tetramethyl rhodamine label n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli-m15 Escherichia coli M15 n/a 1571690 unspecified role unspecified role neutral component neutral component n-terminus 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 false O95259-2:p.Leu154Asn mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false O95259-2:p.[Phe714Ser;Phe717Ser] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false O95259-2:p.[Leu674Asn;Ile678Asn;Phe714Ser;Phe717Ser] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false O95259-2:p.[Arg677Asn;Arg681Asn;Phe714Ser;Phe717Ser] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false O95259-2:p.[Arg675Asn;Arg677Asn;Arg681_Lys682delinsAsnAsn;Phe714Ser;Phe717Ser] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false O95259-2:p.[Phe151Asn;Leu154Asn] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false O95259-2:p.[Phe151Ser;Leu154Ser;Leu158Ser] mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction false false false 1571685 Table 1 Calcium. The interaction was only observed in the presence of Ca2+ (25 micromolar). In the absence of Ca2+, no or very weak (10-fold increased Kd values) interactions were detected. Different hEAG1 constructs interacted with hCaM: hEAG1(1-206) (Kd = 184 +/- 36 x 10E-9 molar), hEAG1(649-867) (Kd = 116 +/- 44 x 10E-9 molar), hEAG1(674-867) (Kd = 127 +/- 15 x 10E-9 molar), hEAG1(684-867) (Kd = 136 +/- 19 x 10E-9 molar). For hEAG1(649-700), hEAG1(479-673) and hEAG1(771-962) no binding was detected (Kd > 5000 x 10E-9 molar). In the hEAG1(1-206) construct, the leu154asn (Kd = 577 +/- 75 x 10E-9 molar) and phe151asn_leu154asn (Kd = 4560 +/- 593 x 10E-9 molar) mutations decrease binding, while the phe151ser_leu154ser_leu158ser (Kd > 5000 x 10E-9 molar) mutation disrupts binding. The phe714ser_phe717ser mutation decreases binding of the hEAG1(674-867) (to Kd = 735 +/- 114 x 10E-9 molar) and hEAG1(649-867) (to Kd = 661 +/- 117 x 10E-9 molar) constructs. In the hEAG1(649-867) construct, the arg675asn_arg677asn_arg681asn_lys682asn mutant had little effect (Kd = 146 +/- 11 x 10E-9 molar), while the leu674asn_ile678asn_phe714ser_phe717ser (Kd = 714 +/- 52 x 10E-9 molar), arg677asn_arg681asn_phe714ser_phe717ser (Kd = 1340 +/- 241 x 10E-9 molar), and arg675asn_arg677asn_arg681asn_lys682asn_phe714ser_phe717ser (Kd = 1960 +/- 335 x 10E-9 molar) mutations decreased binding. calm1-kcnh1-3 1571685 n/a 1571689 unspecified role unspecified role neutral component neutral component 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 false n-terminus tetramethyl rhodamine label tetramethyl rhodamine label n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli-m15 Escherichia coli M15 n/a 1571690 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false 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 false n-terminus mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction false false false 1571685 Table 1 Calcium. The interaction was only observed in the presence of Ca2+ (25 micromolar). In the absence of Ca2+, binding was not detected. calm1-kcnh1-4 1571685 n/a 1571690 unspecified role unspecified role neutral component neutral component n-terminus tetramethyl rhodamine label tetramethyl rhodamine label n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false O95259-2:p.Pro706Cys mutation mutation certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) n/a 1571689 unspecified role unspecified role neutral component neutral component 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 false ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction false false false 1571685 Fig. 3a-c Calcium. The interaction was only observed in the presence of Ca2+. In the absence of Ca2+, binding was not detected. In the presence of Ca2+ hCaM bound the hEAG1 peptide (residues 145-165) with Kd = 45 +/- 9 x 10E-9 molar and the hEAG1 peptide (residues 706-725) with Kd = 223 +/- 44 x 10E-9 molar. hCaM did not bind TMR-labeled hEAG1 peptide (residues 670-688). Reinvestigation with Cy5-labeled hEAG1 peptide (residues 670-688) showed binding to hCaM with Kd = 327 +/- 112 x 10E-9 molar in the presence of Ca2+. calm1-kcnh1-6 1571685 n/a 1571690 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 n-terminus 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 false ecobb Escherichia coli (strain B / BL21) n/a 1571689 unspecified role unspecified role neutral component neutral component 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 false n-terminus bodipy label bodipy label n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli-m15 Escherichia coli M15 direct interaction direct interaction false false false 1571685 Calcium. The interaction was only observed in the presence of Ca2+ (25 micromolar). In the absence of Ca2+, binding was not detected. calm1-kcnh1-7 1571685 n/a 1571689 unspecified role unspecified role neutral component neutral component 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 false ecobb Escherichia coli (strain B / BL21) n/a 1571690 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 n-terminus cy5 label cy5 label n-term range n-terminal range n-term range n-term range n-terminal range n-term range false O95259-2:p.Leu670Cys mutation mutation certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) direct interaction direct interaction false false false 1571685 The interaction was measured in the presence of calcium (Ca2+). kcnh1-calm1 1571686 n/a 1571690 unspecified role unspecified role bait bait n_terminus 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 false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region 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) n/a 1571689 unspecified role unspecified role prey prey 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 false ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction false false false Fig. 4 Calcium. The interaction was only observed in the presence of Ca2+ (25 micromolar). In the absence of Ca2+, no interaction was detected. P62158 decreases activity of O95259 CHEBI:29108 increases binding of P62158 to O95259 kcnh1-calm1-1 1571687 n/a 1571689 unspecified role unspecified role prey prey 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 false region bodipy label bodipy label undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecoli-m15 Escherichia coli M15 n/a 1571690 unspecified role unspecified role bait bait region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) direct interaction direct interaction false false false Fig. 2, Fig. 3a-c calm1-kcnh1-5 1571688 n/a 1571690 unspecified role unspecified role prey prey n-terminus mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false 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 false ecobb Escherichia coli (strain B / BL21) n/a 1571689 unspecified role unspecified role bait bait 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 false ecoli-m15 Escherichia coli M15 direct interaction direct interaction false false false 1571688 Temp = 20C Calcium. The interaction was only observed in the presence of Ca2+ (25 micromolar). In the absence of Ca2+, no specific binding was detected.