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/ baekgaard-2006-2 The plant plasma membrane Ca2+ pump ACA8 contains overlapping as well as physically separated autoinhibitory and calmodulin-binding domains. in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Baekgaard L., Luoni L., De Michelis MI., Palmgren MG. BioCreative - Critical Assessment of Information Extraction systems in Biology palmgren@kvl.dk J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation baekgaard-2006-1 The plant plasma membrane Ca2+ pump ACA8 contains overlapping as well as physically separated autoinhibitory and calmodulin-binding domains. in vitro In vitro far western blotting far western blotting Affinity blotting Affinity blotting predetermined predetermined participant predetermined Baekgaard L., Luoni L., De Michelis MI., Palmgren MG. BioCreative - Critical Assessment of Information Extraction systems in Biology palmgren@kvl.dk J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation aca8_arath Calcium-transporting ATPase 8, plasma membrane-type Ca(2+)-ATPase isoform 8 ACA8 At5g57110 MUL3.5 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MTSLLKSSPGRRRGGDVESGKSEHADSDSDTFYIPSKNASIERLQQWRKAALVLNASRRFRYTLDLKKEQETREMRQKIRSHAHALLAANRFMDMGRESGVEKTTGPATPAGDFGITPEQLVIMSKDHNSGALEQYGGTQGLANLLKTNPEKGISGDDDDLLKRKTIYGSNTYPRKKGKGFLRFLWDACHDLTLIILMVAAVASLALGIKTEGIKEGWYDGGSIAFAVILVIVVTAVSDYKQSLQFQNLNDEKRNIHLEVLRGGRRVEISIYDIVVGDVIPLNIGNQVPADGVLISGHSLALDESSMTGESKIVNKDANKDPFLMSGCKVADGNGSMLVTGVGVNTEWGLLMASISEDNGEETPLQVRLNGVATFIGSIGLAVAAAVLVILLTRYFTGHTKDNNGGPQFVKGKTKVGHVIDDVVKVLTVAVTIVVVAVPEGLPLAVTLTLAYSMRKMMADKALVRRLSACETMGSATTICSDKTGTLTLNQMTVVESYAGGKKTDTEQLPATITSLVVEGISQNTTGSIFVPEGGGDLEYSGSPTEKAILGWGVKLGMNFETARSQSSILHAFPFNSEKKRGGVAVKTADGEVHVHWKGASEIVLASCRSYIDEDGNVAPMTDDKASFFKNGINDMAGRTLRCVALAFRTYEAEKVPTGEELSKWVLPEDDLILLAIVGIKDPCRPGVKDSVVLCQNAGVKVRMVTGDNVQTARAIALECGILSSDADLSEPTLIEGKSFREMTDAERDKISDKISVMGRSSPNDKLLLVQSLRRQGHVVAVTGDGTNDAPALHEADIGLAMGIAGTEVAKESSDIIILDDNFASVVKVVRWGRSVYANIQKFIQFQLTVNVAALVINVVAAISSGDVPLTAVQLLWVNLIMDTLGALALATEPPTDHLMGRPPVGRKEPLITNIMWRNLLIQAIYQVSVLLTLNFRGISILGLEHEVHEHATRVKNTIIFNAFVLCQAFNEFNARKPDEKNIFKGVIKNRLFMGIIVITLVLQVIIVEFLGKFASTTKLNWKQWLICVGIGVISWPLALVGKFIPVPAAPISNKLKVLKFWGKKKNSSGEGSL calm_bovin Calmodulin CALM CAM protein protein bovin Bos taurus (Bovine) Bovine MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK aca8-cam-1 Surface plasmon resonance reveals interaction between ACA8 and CAM proteins 503000 N/A 503002 unspecified role unspecified role bait bait n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Trp47Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 503003 unspecified role unspecified role prey prey direct interaction direct interaction false false false kinetic constants could not be determined because signal was too low. Figure 6 Table 2 aca8-cam-2 SPR analysis reveals interaction between ACA8 and CAM proteins 503000 N/A 503002 unspecified role unspecified role bait bait n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Arg48Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 503003 unspecified role unspecified role prey prey direct interaction direct interaction false false false Figure 6 Table 2 Kd = 158.0 SE = 4.1 aca8-cam-3 SPR analysis reveals interaction between ACA8 and CAM proteins 503000 N/A 503002 unspecified role unspecified role bait bait Q9LF79:p.Leu44Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 503003 unspecified role unspecified role prey prey direct interaction direct interaction false false false Figure 6 Table 2 Kd = 46.4 SE = 0.59 aca8-cam-4 SPR analysis reveals interaction between ACA8 and CAM proteins 503000 N/A 503003 unspecified role unspecified role prey prey N/A 503002 unspecified role unspecified role bait bait n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Ala51Ser mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Figure 6 Table 2 Kd = 45.0 SE = 0.8 aca8-cam-5 SPR analysis reveals interaction between ACA8 and CAM proteins 503000 N/A 503002 unspecified role unspecified role bait bait Q9LF79:p.Val53Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 503003 unspecified role unspecified role prey prey direct interaction direct interaction false false false Figure 6 Table 2 Kd = 65.1 SE = 1.17 aca8-cam-6 SPR analysis reveals interaction between ACA8 and CAM proteins 503000 N/A 503002 unspecified role unspecified role bait bait Q9LF79:p.Ala56Ser mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 503003 unspecified role unspecified role prey prey direct interaction direct interaction false false false Figure 6 Table 2 Kd = 268.1 SE = 7.92 aca8-cam-7 SPR analysis reveals interaction between ACA8 and CAM proteins 503000 N/A 503003 unspecified role unspecified role prey prey N/A 503002 unspecified role unspecified role bait bait Q9LF79:p.Arg58Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Figure 6 Table 2 Kd = 57.8 SE = 0.82 aca8-cam-8 SPR analysis reveals interaction between ACA8 and CAM proteins 503000 N/A 503003 unspecified role unspecified role prey prey N/A 503002 unspecified role unspecified role bait bait n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Phe60Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Figure 6 Table 2 Kinetic constants could not be determined because signal was too low aca8-cam-9 SPR analysis reveals interaction between ACA8 and CAM proteins 503000 N/A 503003 unspecified role unspecified role prey prey N/A 503002 unspecified role unspecified role bait bait n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Arg61Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Figure 6 Table 2 Kd = 49.9 SE = 1.09 aca8-cam-10 SPR analysis reveals interaction between ACA8 and CAM proteins 503000 N/A 503002 unspecified role unspecified role bait bait Q9LF79:p.Leu64Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 503003 unspecified role unspecified role prey prey direct interaction direct interaction false false false Figure 6 Table 2 Kd = 79,5 SE = 2.95 aca8-cam-11 SPR analysis reveals interaction between ACA8 and CAM proteins 503000 N/A 503002 unspecified role unspecified role bait bait Q9LF79:p.Lys67Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 503003 unspecified role unspecified role prey prey direct interaction direct interaction false false false Figure 6 Table 2 Kd = 55.7 SE = 0.95 aca8-cam-12 SPR analysis reveals interaction between ACA8 and CAM proteins 503000 N/A 503002 unspecified role unspecified role bait bait Q9LF79:p.Lys68Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 503003 unspecified role unspecified role prey prey direct interaction direct interaction false false false Figure 6 Table 2 Kd = 58.0 SE = 0.86 aca8-cam-13 SPR analysis reveals interaction between ACA8 and CAM proteins 503000 N/A 503002 unspecified role unspecified role bait bait Q9LF79:p.Leu54Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 503003 unspecified role unspecified role prey prey direct interaction direct interaction false false false Figure 6 Table 2 Kd = 51.3 SE = 0.63 aca8-cam CaM overlay assay reveals interaction between ACA8 and CAM proteins 503001 N/A 503003 unspecified role unspecified role prey prey N/A 503002 unspecified role unspecified role bait bait region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Trp47Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Arg48Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Ala56Ser mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Phe60Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Ala51Ser mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Val53Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Leu54Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Arg58Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Arg61Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Leu64Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Lys67Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q9LF79:p.Lys68Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false The authors state that mutations try47ala, arg48ala, ala56ser and phe60ala severly reduced binding of bovine calmodulin (P62157) to ACA8 protein (Q9LF79) . Whereas the remaining muations only reduced inetraction between the two proteins. Figure 5 Interactions occured in the presence of 1mM calcium chloride