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/ Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase. Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase. J. Biol. Chem. (0021-9258) 2006 imex curation Snider J., Gutsche I., Lin M., Baby S., Cox B., Butland G., Greenblatt J., Emili A., Houry WA. size exclusion chromatography analysis was used. Note also added is the LdcC complex. gutsche@embl-grenoble.fr (regarding EM methodology) walid.houry@utoronto.ca 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined Snider J., Gutsche I., Lin M., Baby S., Cox B., Butland G., Greenblatt J., Emili A., Houry WA. size exclusion chromatography analysis was used. Note also added is the LdcC complex. gutsche@embl-grenoble.fr (regarding EM methodology) gutsche@embl-grenoble.fr (regarding EM methodology) Accepted 2006-OCT-13 by DAVET. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase. Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase. J. Biol. Chem. (0021-9258) 2006 imex curation Snider J., Gutsche I., Lin M., Baby S., Cox B., Butland G., Greenblatt J., Emili A., Houry WA. size exclusion chromatography analysis was used. Note also added is the LdcC complex. gutsche@embl-grenoble.fr (regarding EM methodology) walid.houry@utoronto.ca 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions ecoli Escherichia coli (strain K12) tap tandem affinity purification TAP TAP tap ms participant Identification by mass spectrometry ms participant Snider J., Gutsche I., Lin M., Baby S., Cox B., Butland G., Greenblatt J., Emili A., Houry WA. size exclusion chromatography analysis was used. Note also added is the LdcC complex. gutsche@embl-grenoble.fr (regarding EM methodology) gutsche@embl-grenoble.fr (regarding EM methodology) Accepted 2006-OCT-13 by DAVET. 24-JAN-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase. Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase. J. Biol. Chem. (0021-9258) 2006 imex curation Snider J., Gutsche I., Lin M., Baby S., Cox B., Butland G., Greenblatt J., Emili A., Houry WA. size exclusion chromatography analysis was used. Note also added is the LdcC complex. gutsche@embl-grenoble.fr (regarding EM methodology) walid.houry@utoronto.ca 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro density sedimentation cosedimentation through density gradient density sedimentation predetermined predetermined participant predetermined Snider J., Gutsche I., Lin M., Baby S., Cox B., Butland G., Greenblatt J., Emili A., Houry WA. size exclusion chromatography analysis was used. Note also added is the LdcC complex. gutsche@embl-grenoble.fr (regarding EM methodology) gutsche@embl-grenoble.fr (regarding EM methodology) Accepted 2006-OCT-13 by DAVET. 24-JAN-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase. Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase. J. Biol. Chem. (0021-9258) 2006 imex curation Snider J., Gutsche I., Lin M., Baby S., Cox B., Butland G., Greenblatt J., Emili A., Houry WA. size exclusion chromatography analysis was used. Note also added is the LdcC complex. gutsche@embl-grenoble.fr (regarding EM methodology) walid.houry@utoronto.ca 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro electron microscopy electron microscopy Electron crystallography Electron cryomicroscopy Electron crystallography Electron cryomicroscopy predetermined predetermined participant predetermined Snider J., Gutsche I., Lin M., Baby S., Cox B., Butland G., Greenblatt J., Emili A., Houry WA. size exclusion chromatography analysis was used. Note also added is the LdcC complex. gutsche@embl-grenoble.fr (regarding EM methodology) gutsche@embl-grenoble.fr (regarding EM methodology) Accepted 2006-OCT-13 by DAVET. 24-JAN-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation ldcc_ecoli Constitutive lysine decarboxylase ldcH b0186 ldcC ldc JW0181 protein protein ecoli Escherichia coli (strain K12) MNIIAIMGPHGVFYKDEPIKELESALVAQGFQIIWPQNSVDLLKFIEHNPRICGVIFDWDEYSLDLCSDINQLNEYLPLYAFINTHSTMDVSVQDMRMALWFFEYALGQAEDIAIRMRQYTDEYLDNITPPFTKALFTYVKERKYTFCTPGHMGGTAYQKSPVGCLFYDFFGGNTLKADVSISVTELGSLLDHTGPHLEAEEYIARTFGAEQSYIVTNGTSTSNKIVGMYAAPSGSTLLIDRNCHKSLAHLLMMNDVVPVWLKPTRNALGILGGIPRREFTRDSIEEKVAATTQAQWPVHAVITNSTYDGLLYNTDWIKQTLDVPSIHFDSAWVPYTHFHPIYQGKSGMSGERVAGKVIFETQSTHKMLAALSQASLIHIKGEYDEEAFNEAFMMHTTTSPSYPIVASVETAAAMLRGNPGKRLINRSVERALHFRKEVQRLREESDGWFFDIWQPPQVDEAECWPVAPGEQWHGFNDADADHMFLDPVKVTILTPGMDEQGNMSEEGIPAALVAKFLDERGIVVEKTGPYNLLFLFSIGIDKTKAMGLLRGLTEFKRSYDLNLRIKNMLPDLYAEDPDFYRNMRIQDLAQGIHKLIRKHDLPGLMLRAFDTLPEMIMTPHQAWQRQIKGEVETIALEQLVGRVSANMILPYPPGVPLLMPGEMLTKESRTVLDFLLMLCSVGQHYPGFETDIHGAKQDEDGVYRVRVLKMAG DIP protein P52095 original sequence version: null 4532C6069744ABDF ldci_ecoli Inducible lysine decarboxylase cadA b4131 ldcI JW4092 protein protein ecoli Escherichia coli (strain K12) MNVIAILNHMGVYFKEEPIRELHRALERLNFQIVYPNDRDDLLKLIENNARLCGVIFDWDKYNLELCEEISKMNENLPLYAFANTYSTLDVSLNDLRLQISFFEYALGAAEDIANKIKQTTDEYINTILPPLTKALFKYVREGKYTFCTPGHMGGTAFQKSPVGSLFYDFFGPNTMKSDISISVSELGSLLDHSGPHKEAEQYIARVFNADRSYMVTNGTSTANKIVGMYSAPAGSTILIDRNCHKSLTHLMMMSDVTPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVHAVITNSTYDGLLYNTDFIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMSGGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTSPHYGIVASTETAAAMMKGNAGKRLINGSIERAIKFRKEIKRLRTESDGWFFDVWQPDHIDTTECWPLRSDSTWHGFKNIDNEHMYLDPIKVTLLTPGMEKDGTMSDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDKTKALSLLRALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRIQELAQNIHKLIVHHNLPDLMYRAFEVLPTMVMTPYAAFQKELHGMTEEVYLDEMVGRINANMILPYPPGVPLVMPGEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQADGRYTVKVLKEESKK DIP protein P0A9H3 original sequence version: 58 This sequence has been withdrawn from Uniprot. B9776622A0AE0190 rava_ecoli ATPase RavA yieN Regulatory ATPase variant A ravA b3746 JW3725 protein protein ecoli Escherichia coli (strain K12) MAHPHLLAERISRLSSSLEKGLYERSHAIRLCLLAALSGESVFLLGPPGIAKSLIARRLKFAFQNARAFEYLMTRFSTPEEVFGPLSIQALKDEGRYERLTSGYLPEAEIVFLDEIWKAGPAILNTLLTAINERQFRNGAHVEKIPMRLLVAASNELPEADSSLEALYDRMLIRLWLDKVQDKANFRSMLTSQQDENDNPVPDALQVTDEEYERWQKEIGEITLPDHVFELIFMLRQQLDKLPDAPYVSDRRWKKAIRLLQASAFFSGRSAVAPVDLILLKDCLWYDAQSLNLIQQQIDVLMTGHAWQQQGMLTRLGAIVQRHLQLQQQQSDKTALTVIRLGGIFSRRQQYQLPVNVTASTLTLLLQKPLKLHDMEVVHISFERSALEQWLSKGGEIRGKLNGIGFAQKLNLEVDSAQHLVVRDVSLQGSTLALPGSSAEGLPGEIKQQLEELESDWRKQHALFSEQQKCLFIPGDWLGRIEASLQDVGAQIRQAQQC DIP protein P31473 original sequence version: 98 DIP protein P31473 original sequence version: null 3D2EDCAA17240BE9 rava-rava 1 7 unspecified role unspecified role neutral component neutral component ecobb Escherichia coli (strain B / BL21) physical association physical association Size exclusion chromatography analysis using 3 M RavA revealed that the protein forms oligomeric species and that this oligomerization is enhanced in the presence of nucleotide (Fig. 5A). The largest species formed runs near the 443-kDa marker. 5 A The oligomerisation was enhanced in the presence of ATP. rava-rava-1 3 7 unspecified role unspecified role neutral component neutral component ecobb Escherichia coli (strain B / BL21) physical association physical association analytical ultracentrifugation sedimentation equilibrium studies were performed in the presence of ATP. The best fit to the data set corresponds to a monodisperse species of 310 kDa in size (Fig. 5B). This molecular weight corresponds closely with that expected of a hexameric species. 5 B The experiment was performed in the presence of ATP, as ATP enhances oligomerisation. The best fit to the data set corresponds to a monodisperse species of about 10 kDa in size (Fig. 5B). This molecular weight corresponds closely with that expected of a hexameric species. Thus they propose that the functional state of RavA is a hexameric oligomer in the presence of nucleotides. rava-rava-2 4 7 unspecified role unspecified role neutral component neutral component ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction THe species circled appear to be hexameric. analysis of RavA by electron microscopy. Shown are negatively stained RavA particles in the presence of ADP. Some particles that could be attributed to hexamers are circled. 5 C A range of different particles were seen, which were thought to correspond to a range of oligomeric states. On Fig. 5 C, those corresponding to hexamers are circled. rava-cada 2 6 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role bait bait c-terminal tap tagged calmodulin binding peptide plus protein a tag CBP-ProtA tagged CBP-ProtA tagged tap tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range ecoli Escherichia coli (strain K12) physical association physical association pull-down of endogenous, C-terminally TAP-tagged RavA. Identification of the bands by mass spectrometry reveals that RavA interacts with the inducible lysine decarboxylase enzyme, LdcI 6 A rava-rava-3 1 7 unspecified role unspecified role neutral component neutral component ecobb Escherichia coli (strain B / BL21) physical association physical association 6 B, panel 1 18 M RavA protein migrates as a single species with an apparent molecular mass of 440 kDa representing the hexameric species This was carried out in the presence of ATP, which enhances oligomerisation of RavA. rava-cada-1 1 7 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association A mixture of 10 M LdcI with excess RavA (18 M) results in a large shift in the migration of both species indicating a strong interaction (Fig. 6B, panel 3). A range of higher order oligomers are formed, with the predominant complex (boxed) migrating at or near the void volume of the column, suggesting a size of 50 MDa or greater. 6 B, panel 3 A range of higher order oligomers are formed, with the predominant complex (boxed) migrating at or near the void volume of the column, suggesting a size of approx. 50 MDa or greater. This was carried out in the presence of ATP, which enhances oligomerisation of RavA. rava-cada-2 1 6 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association The predominant complex formed changes if 18 M RavA is incubated with 2-fold excess of LdcI (40 M). 6 B, panel 4 The predominant complex formed changes if 18 uM RavA is incubated with approx. 2-fold excess of LdcI (40 uM). The formed complex elutes within the column volume, and its molecular mass is 3�5 MDa as estimated by sedimentation equilibrium experiments (Fig. 6B, left box in panel 4 and data not shown). They term this complex the fraction three complex (FTC). rava-cada-3 1 7 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association LdcI binds to 3 M RavA in the absence and presence of ATP(Fig. 6C, panels 4 and 5), however, the size of the complex is smaller in the absence of ATP as compared with when ATP is present. 6 C panel 4 LdcI binds to RavA in the absence and presence of ATP(Fig. 6C, panels 4 and 5), however, the size of the complex is smaller in the absence of ATP as compared with when ATP is present. These data indicate that LdcI probably does not promote the oligomerization of RavA in the absence of nucleotide. In Fig. 6C (panel 5), the FTC is also observed. cada-cada 4 6 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) direct interaction direct interaction A 20 � resolution structure of the D5-symmetric LdcI oligomer (Fig. 7, A�C) was obtained as described under �Materials and Methods.� The existence of a 5-fold symmetry axis in the particle was corroborated by multivariate statistical analysis. Fig. 7B shows the pronounced 5-fold symmetrical shape of the top-view class average and the 2-fold symmetry of the side-view class average. Some dimeric dissociation products could always be detected in the background of the micrographs (Fig. 7A), 7 A-C Some dimeric dissociation products could always be detected in the background of the micrographs (Fig. 7A), The random orientation of single particles on the carbon film made this specimen particularly suitable for a three-dimensional reconstruction by angular reconstitution approach (Fig. 7C). The decamer of LdcI revealed itself as two pentameric rings, tightly stacked together back-to-back and turned ~35C with respect to each other. Each monomer has a globular shape. The relative rotation of the two rings confers a pronounced handedness to the molecule responsible for the "plait-like" appearance of the side view. rava-cada-4 4 6 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) physical association physical association at 2-fold excess of RavA (3.24 M RavA and 1.8 M LdcI in the presence of 3 mM ADP or ATP S), the background became clean, individual LdcI views virtually disappeared, and most of the protein was present as an amazingly large and apparently homogeneous RavA LdcI complex. 7 D-F Analysis of the data revealed that the ADP-RavA-Ldc1 complex contained not only one, but two LdcI decamers sandwiched together and glued by additional densities that certainly arise from RavA (Fig. 7D). This was carried out in the presence of ADP, which enhances the oligomerisation of RavA. cada-cada-1 1 6 unspecified role unspecified role neutral component neutral component ecobb Escherichia coli (strain B / BL21) physical association physical association 10 M LdcI (Fig. 6B, panel 2) also elutes as a single species, with an apparent molecular mass of 600 kDa probably representing the decameric species that is known to be formed by LdcI 6 B, panel 2, 6 C, panel 1 The size of the LdcI oligomer (decamer) is unaffected by the presence or absence of ATP. ldcc-ldcc 1 5 unspecified role unspecified role neutral component neutral component ecobb Escherichia coli (strain B / BL21) physical association physical association 6, panel 5 LdcC is also a PLP-dependent enzyme that decarboxylates lysine and possibly assembles into a decamer LdcC may form decameric complexes.