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/ mapelli-2007-1 The mad2 conformational dimer: structure and implications for the spindle assembly checkpoint. in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined Mapelli M., Massimiliano L., Santaguida S., Musacchio A. andrea.musacchio@ifom-ieo-campus.it Cell (0092-8674) 2007 Only protein-protein interactions 5 imex curation mapelli-2007-3 The mad2 conformational dimer: structure and implications for the spindle assembly checkpoint. in vitro In vitro pull down pull down predetermined predetermined participant predetermined Mapelli M., Massimiliano L., Santaguida S., Musacchio A. andrea.musacchio@ifom-ieo-campus.it Cell (0092-8674) 2007 Only protein-protein interactions 5 imex curation mapelli-2007-2 The mad2 conformational dimer: structure and implications for the spindle assembly checkpoint. in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Mapelli M., Massimiliano L., Santaguida S., Musacchio A. andrea.musacchio@ifom-ieo-campus.it Cell (0092-8674) 2007 Only protein-protein interactions 5 imex curation mad2 peptide MAD2 binding peptide 1 peptide peptide polypeptide oligopeptide oligopeptide polypeptide in vitro In vitro SWYSYPPPQRAV An amino acid sequence which is not represented in UniProt md2l1_human Mitotic spindle assembly checkpoint protein MAD2A MAD2L1 MAD2 Mitotic arrest deficient 2-like protein 1 protein protein human Homo sapiens Human MALQLSREQGITLRGSAEIVAEFFSFGINSILYQRGIYPSETFTRVQKYGLTLLVTTDLELIKYLNNVVEQLKDWLYKCSVQKLVVVISNIESGEVLERWQFDIECDKTAKDDSAPREKSQKAIQDEIRSVIRQITATVTFLPLLEVSCSFDLLIYTDKDLVVPEKWEESGPQFITNSEEVRLRSFTTTIHKVNSMVAYKIPVND cdc20_human Cell division cycle protein 20 homolog CDC20 p55CDC protein protein human Homo sapiens Human MAQFAFESDLHSLLQLDAPIPNAPPARWQRKAKEAAGPAPSPMRAANRSHSAGRTPGRTPGKSSSKVQTTPSKPGGDRYIPHRSAAQMEVASFLLSKENQPENSQTPTKKEHQKAWALNLNGFDVEEAKILRLSGKPQNAPEGYQNRLKVLYSQKATPGSSRKTCRYIPSLPDRILDAPEIRNDYYLNLVDWSSGNVLAVALDNSVYLWSASSGDILQLLQMEQPGEYISSVAWIKEGNYLAVGTSSAEVQLWDVQQQKRLRNMTSHSARVGSLSWNSYILSSGSRSGHIHHHDVRVAEHHVATLSGHSQEVCGLRWAPDGRHLASGGNDNLVNVWPSAPGEGGWVPLQTFTQHQGAVKAVAWCPWQSNVLATGGGTSDRHIRIWNVCSGACLSAVDAHSQVCSILWSPHYKELISGHGFAQNQLVIWKYPTMAKVAELKGHTSRVLSLTMSPDGATVASAAADETLRLWRCFELDPARRREREKASAAKSSLIHQGIR mad2l1-mad2l1-1 The MAD2 dimer stabilized by a synthetic peptide is isolated by gel filtration 682471 N/A 682474 unspecified role unspecified role neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) N/A 682475 unspecified role unspecified role neutral component neutral component mutated region TAKDDSAPR -> GSG experimental feature experimental feature substitut analysis substitution analysis substitut analysis certain certain sequence position certain certain certain sequence position certain false ecobb Escherichia coli (strain B / BL21) Stoichiometry: 1.0 N/A 682475 unspecified role unspecified role neutral component neutral component ecobb Escherichia coli (strain B / BL21) Stoichiometry: 1.0 physical association physical association false false false Figure 1A mad2l1-mad2l1-3 The MAD2 dimer is isolated by gel filtration 682471 N/A 682475 unspecified role unspecified role neutral component neutral component Q13257:p.Leu13Ala closed conformation mutation increasing mutation increasing interaction mutation increasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false ecobb Escherichia coli (strain B / BL21) N/A 682475 unspecified role unspecified role neutral component neutral component ecobb Escherichia coli (strain B / BL21) physical association physical association false false false Supp. Fig 2 cdc20-mad2l1-1 Interaction between MAD2L1 and CDC20 identified by GST pulldown 682472 N/A 682475 unspecified role unspecified role prey prey Q13257:p.Val193Gln mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain false ecobb Escherichia coli (strain B / BL21) N/A 682476 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 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) direct interaction direct interaction false false false Fig. 2F, 3B, 4D Loss of residues 196-205 force Mad2 into a closed conformation cdc20-mad2l1-2 Interaction between the MAD2L1 dimer and CDC20 identified by GST pulldown 682472 N/A 682475 unspecified role unspecified role prey prey ecobb Escherichia coli (strain B / BL21) N/A 682476 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 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 682475 unspecified role unspecified role prey prey region open conformation 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) physical association physical association false false false Figs. 2F, 4D, 5E Phe141 and Arg184 are required for the interaction of wild type Mad2 (C-MAD2) with Mad2 locked in the open conformation (O-MAD2), the same mutations in O-Mad2 did not significantly alter the binding to C-Mad2. Val193 generates a mutant that is locked as O-Mad2 and is totally defective in Cdc20 binding. Mutating Trp100 of Mad2 to Tyr (W100Y) generates a stable O-Mad2 mutant that binds to GST-Cdc20111�138 with very slow kinetics. mad2l1-mad2l1-2 The MAD2 dimer stabilized by a synthetic peptide crystal structure 682473 N/A 682475 unspecified role unspecified role neutral component neutral component mutated region TAKDDSAPR -> GSG experimental feature experimental feature substitut analysis substitution analysis substitut analysis certain certain sequence position certain certain certain sequence position certain false ecobb Escherichia coli (strain B / BL21) N/A 682475 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 ecobb Escherichia coli (strain B / BL21) N/A 682474 unspecified role unspecified role neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association false false false Table 1 2.9 A Free = 27.4%