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/ mastny-2013-3 CtpB assembles a gated protease tunnel regulating cell-cell signaling during spore formation in Bacillus subtilis. in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Mastny M., Heuck A., Kurzbauer R., Heiduk A., Boisguerin P., Volkmer R., Ehrmann M., Rodrigues C.D., Rudner D.Z., Clausen T. clausen@imp.univie.ac.at Cell (0092-8674) 2013 Only protein-protein interactions imex curation mastny-2013-1 CtpB assembles a gated protease tunnel regulating cell-cell signaling during spore formation in Bacillus subtilis. in vitro In vitro protease assay protease assay weight by comassie molecular weight estimation by coomasie staining weight by comassie Mastny M., Heuck A., Kurzbauer R., Heiduk A., Boisguerin P., Volkmer R., Ehrmann M., Rodrigues C.D., Rudner D.Z., Clausen T. clausen@imp.univie.ac.at Cell (0092-8674) 2013 Only protein-protein interactions imex curation mastny-2013-5 CtpB assembles a gated protease tunnel regulating cell-cell signaling during spore formation in Bacillus subtilis. in vitro In vitro itc isothermal titration calorimetry ITC ITC itc predetermined predetermined participant predetermined Mastny M., Heuck A., Kurzbauer R., Heiduk A., Boisguerin P., Volkmer R., Ehrmann M., Rodrigues C.D., Rudner D.Z., Clausen T. clausen@imp.univie.ac.at Cell (0092-8674) 2013 Only protein-protein interactions imex curation mastny-2013-2 CtpB assembles a gated protease tunnel regulating cell-cell signaling during spore formation in Bacillus subtilis. in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column weight by comassie molecular weight estimation by coomasie staining weight by comassie Mastny M., Heuck A., Kurzbauer R., Heiduk A., Boisguerin P., Volkmer R., Ehrmann M., Rodrigues C.D., Rudner D.Z., Clausen T. clausen@imp.univie.ac.at Cell (0092-8674) 2013 Only protein-protein interactions imex curation mastny-2013-4 CtpB assembles a gated protease tunnel regulating cell-cell signaling during spore formation in Bacillus subtilis. in vitro In vitro peptide array peptide array antibody detection identification by antibody antibody detection Mastny M., Heuck A., Kurzbauer R., Heiduk A., Boisguerin P., Volkmer R., Ehrmann M., Rodrigues C.D., Rudner D.Z., Clausen T. clausen@imp.univie.ac.at Cell (0092-8674) 2013 Only protein-protein interactions imex curation ctpb_bacsu Carboxy-terminal processing protease CtpB ctpB yvjB BSU35240 protein protein bacsu Bacillus subtilis MNQKIMAVIAAGSMLFGGAGVYAGINLLEMDKPQTAAVPATAQADSERDKAMDKIEKAYELISNEYVEKVDREKLLEGAIQGMLSTLNDPYSVYMDKQTAKQFSDSLDSSFEGIGAEVGMEDGKIIIVSPFKKSPAEKAGLKPNDEIISINGESMAGKDLNHAVLKIRGKKGSSVSMKIQRPGTKKQLSFRIKRAEIPLETVFASEKKVQGHSVGYIAISTFSEHTAEDFAKALRELEKKEIEGLVIDVRGNPGGYLQSVEEILKHFVTKDQPYIQIAERNGDKKRYFSTLTHKKAYPVNVITDKGSASASEILAGALKEAGHYDVVGDTSFGKGTVQQAVPMGDGSNIKLTLYKWLTPNGNWIHKKGIEPTIAIKQPDYFSAGPLQLKEPLKVDMNNEDVKHAQVLLKGLSFDPGREDGYFSKDMKKAVMAFQDQNKLNKTGVIDTRTAETLNQQIEKKKSDEKNDLQLQTALKSLFVN poly alanine peptide peptide peptide polypeptide oligopeptide oligopeptide polypeptide chemical synthesis Chemical synthesis (Chemical synthesis) sp4fa_bacsu Stage IV sporulation protein FA spoIVFA bofB BSU27980 protein protein bacsu Bacillus subtilis MSHRADEIRKRLEKRRKQLSGSKRFSTQTVSEKQKPPSWVMVTDQEKHGTLPVYEDNMPTFNGKHPLVKTDSIILKCLLSACLVLVSAIAYKTNIGPVSQIKPAVAKTFETEFQFASASHWFETKFGNPLAFLAPEHKNKEQQIEVGKDLIAPASGKVQQDFQDNGEGIKVETSSDKIDSVKEGYVVEVSKDSQTGLTVKVQHADNTYSIYGELKDVDVALYDFVDKGKKLGSIKLDDHNKGVYYFAMKDGDKFIDPIQVISFE sp4b_bacsu SpoIVB peptidase spoIVB Sporulation factor IV B protease Stage IV sporulation protein B BSU24230 protein protein bacsu Bacillus subtilis MPDNIRKAVGLILLVSLLSVGLCKPLKEYLLIPTQMRVFETQTQAIETSLSVNAQTSESSEAFTVKKDPHEIKVTGKKSGESELVYDLAGFPIKKTKVHVLPDLKVIPGGQSIGVKLHSVGVLVVGFHQINTSEGKKSPGETAGIEAGDIIIEMNGQKIEKMNDVAPFIQKAGKTGESLDLLIKRDKQKIKTKLIPEKDEGEGKYRIGLYIRDSAAGIGTMTFYEPKTKKYGALGHVISDMDTKKPIVVENGEIVKSTVTSIEKGTGGNPGEKLARFSSERKTIGDINRNSPFGIFGTLHQPIQNNISDQALPVAFSTEVKKGPAEILTVIDDDKVEKFDIEIVSTTPQKFPATKGMVLKITDPRLLKETGGIVQGMSGSPIIQNGKVIGAVTHVFVNDPTSGYGVHIEWMLSEAGIDIYGKEKAS fmllrrkaya FMLLRRKAYA peptide peptide peptide polypeptide oligopeptide oligopeptide polypeptide chemical synthesis Chemical synthesis (Chemical synthesis) FMLLRRKAYA pdz binder NLVLAGLHSYA peptide peptide polypeptide oligopeptide oligopeptide polypeptide chemical synthesis Chemical synthesis (Chemical synthesis) NLVLAGLHSYA ctpb-ctpb 1475905 n/a 1475910 unspecified role unspecified role neutral component neutral component c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false Supp. Table 1 1.9A Rworking = 0.2, Rfree = 0.22 ctpb-ctpb-5 1475905 n/a 1475911 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false chemical synthesis Chemical synthesis (Chemical synthesis) n/a 1475910 unspecified role unspecified role neutral component neutral component c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ser-309 direct binding direct binding region direct binding certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 1475926 1475922 direct interaction direct interaction false false false Supp. Fig. 3 Supp. Table 1 2.6A Rworking = 0.23, Rfree = 0.24 ctpb-ctpb-2 1475905 n/a 1475910 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 c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false O35002:p.Val209Ala mutation mutation certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false Supp. Table 1 1.8A Rworking = 0.18, Rfree = 0.19 ctpb-ctpb-6 1475905 n/a 1475911 unspecified role unspecified role neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) Stoichiometry: 1.0 n/a 1475910 unspecified role unspecified role neutral component neutral component O35002:p.Arg168Ala mutation mutation certain certain sequence position certain certain certain sequence position certain false c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false Supp. Table 1 2.4A Rworking = 0.22, Rfree = 0.26 ctpb-ctpb-4 1475905 n/a 1475910 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 1475910 unspecified role unspecified role neutral component neutral component c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 1475943 1475940 direct interaction direct interaction false false false Supp. Table 1 1.9A Rworking = 0.2, Rfree = 0.23 ctpb-1 1475905 n/a 1475910 unspecified role unspecified role neutral component neutral component c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false O35002:p.Val118Tyr mutation mutation 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 ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false Supp. Table 1. 1.95A Rworking = 0.2, Rfree = 0.24 ctpb-spoivfa 1475906 n/a 1475912 enzyme target enzyme target substrate substrate unspecified role unspecified role 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 extracellular region sufficient to bind sufficient binding region sufficient to bind range range certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1475910 enzyme enzyme unspecified role unspecified role region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain false c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) 1475954 1475952 protein cleavage protein cleavage false false false Fig. 1B, Supp. Fig. 1B, 1D O35002 increases degradation at protein level of P26936 spoivb-spoivfa 1475906 n/a 1475913 enzyme enzyme unspecified role unspecified role 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 ecoli Escherichia coli (strain K12) n/a 1475912 enzyme target enzyme target substrate substrate unspecified role unspecified role extracellular region sufficient to bind sufficient binding region sufficient to bind range range certain certain sequence position certain false 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 ecoli Escherichia coli (strain K12) protein cleavage protein cleavage false false false Fig. 1B P26937 increases degradation at protein level of P26936 In vitro reconstituted cleavage reactions demonstrate that 4B-dependent cleavage of 4FA generates a substrate that can be further digested by CtpB. ctpb-1 1475906 n/a 1475910 putative self putative self putative self putative self 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 O35002:p.Ser309Ala mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate 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 ecoli Escherichia coli (strain K12) protein cleavage protein cleavage false true false Supp. Fig. 1A ctpb-spoivfa-1 1475906 n/a 1475912 enzyme target enzyme target substrate substrate unspecified role unspecified role 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 extracellular region sufficient to bind sufficient binding region sufficient to bind range range range range false ecoli Escherichia coli (strain K12) n/a 1475910 enzyme enzyme unspecified role unspecified role region required to bind necessary binding region required to bind range range certain certain sequence position certain false certain certain sequence position certain range range false c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false O35002:p.Arg168Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false O35002:p.[Val118Tyr;Arg168Phe] mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false O35002:p.Gln338Glu mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain false O35002:p.[Val118Tyr;Arg168Ala] mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false O35002:p.Val118Tyr mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false O35002:p.Arg168Phe mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) protein cleavage protein cleavage false false false Fig. 5C, Sequence length of spoIVFA inferred from information in Fig. 1C. ctpb-fmllrrkaya-2 1475907 n/a 1475914 unspecified role unspecified role neutral component neutral component fmllrrkaya:p.Ala10Val mutation increasing strength mutation increasing interaction strength mutation increasing strength certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) n/a 1475910 unspecified role unspecified role neutral component neutral component O35002:p.Ser309Ala mutation mutation certain certain sequence position certain certain certain sequence position certain false O35002:p.Arg168Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false O35002:p.Val118Tyr mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false O35002:p.Arg168Phe mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false 1475907 Supp. Fig. 4C, 5B The derived dissociation constants (KD) for the full-length CtpB protein were about 3-fold higher than for the isolated PDZ domain ctpb-fmllrrkaya-3 1475907 n/a 1475910 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 c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) n/a 1475914 unspecified role unspecified role neutral component neutral component fmllrrkaya:p.Ala10Val mutation increasing strength mutation increasing interaction strength mutation increasing strength certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) direct interaction direct interaction false false false 1475907 Supp. Fig. 4C The derived dissociation constants (KD) for the full-length CtpB protein were about 3-fold higher than for the isolated PDZ domain ctpb-1 1475908 n/a 1475910 unspecified role unspecified role neutral component neutral component 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 region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false Supp. Fig. 1C pdz_binder-ctpb 1475909 n/a 1475915 unspecified role unspecified role bait bait chemical synthesis Chemical synthesis (Chemical synthesis) n/a 1475910 unspecified role unspecified role prey prey region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain false c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) protein cleavage protein cleavage false false false Fig. 4C, Supp. Fig. 4A, 4B Amino acid subsitutions demonstrated that the PDZ domain of CtpB preferentially binds peptides carrying Tyr/Phe/Trp-Ala/Val combinations at their C terminus