DIP Database of Interacting Proteins 611 Young Drive East; Los Angeles CA 90095; USA http://dip.doe-mbi.ucla.edu/ dip@mbi.ucla.edu 611 Young Drive East; Los Angeles CA 90095; USA http://dip.doe-mbi.ucla.edu/ castro_roa-2013-1 in vitro In vitro protein kinase assay protein kinase assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Castro-Roa D., Garcia-Pino A., De Gieter S., van Nuland NA., Loris R., Zenkin N. DIP original experiment: DIP-266860X Nature chemical biology 2013 IMEx The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu. Castro-Roa D., Garcia-Pino A., De Gieter S., van Nuland NA., Loris R., Zenkin N. Only protein-protein interactions imex curation castro_roa-2013-2 in vitro In vitro saxs x ray scattering saxs predetermined predetermined participant predetermined Castro-Roa D., Garcia-Pino A., De Gieter S., van Nuland NA., Loris R., Zenkin N. DIP original experiment: DIP-266862X Nature chemical biology 2013 IMEx The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu. Castro-Roa D., Garcia-Pino A., De Gieter S., van Nuland NA., Loris R., Zenkin N. Only protein-protein interactions imex curation castro_roa-2013-3 in vitro In vitro nmr nuclear magnetic resonance NMR NMR nmr predetermined predetermined participant predetermined Castro-Roa D., Garcia-Pino A., De Gieter S., van Nuland NA., Loris R., Zenkin N. DIP original experiment: DIP-266863X Nature chemical biology 2013 IMEx The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu. Castro-Roa D., Garcia-Pino A., De Gieter S., van Nuland NA., Loris R., Zenkin N. Only protein-protein interactions imex curation castro_roa-2013-4 in vitro In vitro protein kinase assay protein kinase assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Castro-Roa D., Garcia-Pino A., De Gieter S., van Nuland NA., Loris R., Zenkin N. DIP original experiment: DIP-266861X Nature chemical biology 2013 IMEx The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu. Castro-Roa D., Garcia-Pino A., De Gieter S., van Nuland NA., Loris R., Zenkin N. Only protein-protein interactions imex curation castro_roa-2013-5 in vitro In vitro itc isothermal titration calorimetry ITC ITC itc predetermined predetermined participant predetermined Castro-Roa D., Garcia-Pino A., De Gieter S., van Nuland NA., Loris R., Zenkin N. DIP original experiment: DIP-266864X Nature chemical biology 2013 IMEx The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu. Castro-Roa D., Garcia-Pino A., De Gieter S., van Nuland NA., Loris R., Zenkin N. Only protein-protein interactions imex curation doc_bpp1 Protein kinase doc doc Death on curing protein Toxin doc protein protein bpp1 Enterobacteria phage P1 Bacteriophage P1 MRHISPEELIALHDANISRYGGLPGMSDPGRAEAIIGRVQARVAYEEITDLFEVSATYLVATARGHIFNDANKRTALNSALLFLRRNGVQVFDSPELADLTVGAATGEISVSSVADTLRRLYGSAE eftu1_ecoli Elongation factor Tu 1 tufA b3339 P-43 JW3301 Bacteriophage Q beta RNA-directed RNA polymerase subunit III protein protein ecoli Escherichia coli (strain K12) MSKEKFERTKPHVNVGTIGHVDHGKTTLTAAITTVLAKTYGGAARAFDQIDNAPEEKARGITINTSHVEYDTPTRHYAHVDCPGHADYVKNMITGAAQMDGAILVVAATDGPMPQTREHILLGRQVGVPYIIVFLNKCDMVDDEELLELVEMEVRELLSQYDFPGDDTPIVRGSALKALEGDAEWEAKILELAGFLDSYIPEPERAIDKPFLLPIEDVFSISGRGTVVTGRVERGIIKVGEEVEIVGIKETQKSTCTGVEMFRKLLDEGRAGENVGVLLRGIKREEIERGQVLAKPGTIKPHTKFESEVYILSKDEGGRHTPFFKGYRPQFYFRTTDVTGTIELPEGVEMVMPGDNIKMVVTLIHPIAMDDGLRFAIREGGRTVGAGVVAKVLG doc-tufa-1 3259585 N/A 3259590 enzyme target enzyme target substrate substrate unspecified role unspecified role P0CE47:p.Thr383Val T383V mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate substitut analysis substitution analysis substitut analysis certain certain sequence position certain certain certain sequence position certain false pT383 pT383 opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt modified residue ms mass detection of residue modification modified residue ms certain certain sequence position certain certain certain sequence position certain false ecolx Escherichia coli N/A 3259591 enzyme enzyme unspecified role unspecified role ecolx Escherichia coli phosphorylation phosphorylation reaction phosphorylation false false false doc-tufa-2 3259586 N/A 3259590 unspecified role unspecified role unspecified role unspecified role ecolx Escherichia coli N/A 3259591 unspecified role unspecified role unspecified role unspecified role ecolx Escherichia coli direct interaction direct interaction false false false doc-tufa-3 3259587 N/A 3259590 unspecified role unspecified role unspecified role unspecified role ecolx Escherichia coli N/A 3259591 unspecified role unspecified role unspecified role unspecified role ecolx Escherichia coli direct interaction direct interaction false false false doc-tufa-4 3259588 N/A 3259590 enzyme target enzyme target substrate substrate unspecified role unspecified role ecolx Escherichia coli N/A 3259591 enzyme enzyme unspecified role unspecified role Q06259:p.His66Tyr H66Y mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate substitut analysis substitution analysis substitut analysis certain certain sequence position certain certain certain sequence position certain false ecolx Escherichia coli phosphorylation phosphorylation reaction phosphorylation false false false doc-tufa-5 3259589 N/A 3259590 unspecified role unspecified role unspecified role unspecified role ecolx Escherichia coli N/A 3259591 unspecified role unspecified role unspecified role unspecified role Q06259:p.Arg64Gly R64G mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength substitut analysis substitution analysis substitut analysis certain certain sequence position certain certain certain sequence position certain false ecolx Escherichia coli direct interaction direct interaction false false false 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/ garcia_pino-2010-3 Allostery and intrinsic disorder mediate transcription regulation by conditional cooperativity. in vitro In vitro itc isothermal titration calorimetry ITC ITC itc predetermined predetermined participant predetermined Garcia-Pino A., Balasubramanian S., Wyns L., Gazit E., De Greve H., Magnuson RD., Charlier D., van Nuland NA., Loris R. agarciap@vub.ac.be Cell (0092-8674) 2010 Only protein-protein interactions imex curation garcia_pino-2010-4 Allostery and intrinsic disorder mediate transcription regulation by conditional cooperativity. in vitro In vitro emsa electrophoretic mobility shift assay Gel retardation assay band shift band shift Gel retardation assay emsa weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Garcia-Pino A., Balasubramanian S., Wyns L., Gazit E., De Greve H., Magnuson RD., Charlier D., van Nuland NA., Loris R. agarciap@vub.ac.be Cell (0092-8674) 2010 Only protein-protein interactions imex curation garcia_pino-2010-5 Allostery and intrinsic disorder mediate transcription regulation by conditional cooperativity. in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Garcia-Pino A., Balasubramanian S., Wyns L., Gazit E., De Greve H., Magnuson RD., Charlier D., van Nuland NA., Loris R. agarciap@vub.ac.be Cell (0092-8674) 2010 Only protein-protein interactions imex curation garcia_pino-2010-1 Allostery and intrinsic disorder mediate transcription regulation by conditional cooperativity. in vitro In vitro cd circular dichroism CD CD cd predetermined predetermined participant predetermined Garcia-Pino A., Balasubramanian S., Wyns L., Gazit E., De Greve H., Magnuson RD., Charlier D., van Nuland NA., Loris R. agarciap@vub.ac.be Cell (0092-8674) 2010 Only protein-protein interactions imex curation garcia_pino-2010-2 Allostery and intrinsic disorder mediate transcription regulation by conditional cooperativity. in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Garcia-Pino A., Balasubramanian S., Wyns L., Gazit E., De Greve H., Magnuson RD., Charlier D., van Nuland NA., Loris R. Wild-type Doc was coupled to the CM5 chip using the thiol method (1998) and Phd was injected in concentrations between 100 nM and 250 mM. With this approach the kinetic parameters of the binding to both Phd binding sites can be extracted. agarciap@vub.ac.be Cell (0092-8674) 2010 Only protein-protein interactions imex curation phd_bpp1 Antitoxin phd phd Addiction protein pdh Prevent host death protein protein protein bpp1 Enterobacteria phage P1 Bacteriophage P1 MQSINFRTARGNLSEVLNNVEAGEEVEITRRGREPAVIVSKATFEAYKKAALDAEFASLFDTLDSTNKELVNR ta_p1_operator Operator region of the phd/doc TA operon dna deoxyribonucleic acid DNA deoxyribonucleic acid dna DNA deoxyribonucleic acid bpp1 Enterobacteria phage P1 Bacteriophage P1 Contains two phd binding sites: OR1 consisting of a perfect palindrome (5'-TGTGTACACA-3' and OR2 consisting of an imperfect palindrome (5'-TGAGTACACA-3'). doc_bpp1 Protein kinase doc doc Death on curing protein Toxin doc protein protein bpp1 Enterobacteria phage P1 Bacteriophage P1 MRHISPEELIALHDANISRYGGLPGMSDPGRAEAIIGRVQARVAYEEITDLFEVSATYLVATARGHIFNDANKRTALNSALLFLRRNGVQVFDSPELADLTVGAATGEISVSSVADTLRRLYGSAE phd_c_doc 976548 n/a 976553 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) n/a 976554 unspecified role unspecified role neutral component neutral component c terminal 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) direct interaction direct interaction false false false Fig 2D, Table S4. Temp=25C, pH 7.5. For mutations DocH66Y/A61R, Kd = 0.29 uM, DocH66Y/N78W Kd = 43.9 uM. phd_or1 976548 n/a 976555 unspecified role unspecified role neutral component neutral component n/a 976554 unspecified role unspecified role neutral component neutral component c terminal 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) direct interaction direct interaction false false false Fig 3D, Table S4. Temp=25C, pH 7.5. Binding of Phd to OR1 (a single palindromic sequence (5'-TGTGTACACA-3'))from the phd/doc operon operator. phd_doc_or1 976548 n/a 976553 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) n/a 976555 unspecified role unspecified role neutral component neutral component n/a 976554 unspecified role unspecified role neutral component neutral component c terminal 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) physical association physical association false false false Fig 3E, Table S4. Temp=25C, pH 7.5. Doc-mediated enhancement of Phd binding to operator DNA is observed in operator regions containing a single Phd-binding site;the affinity of Phd2/Doc for OR1 is 0.3 uM, about 10-fold higher than the binding constant of Phd2 for OR1. phd_ta 976549 n/a 976554 unspecified role unspecified role prey prey Q06253:p.Phe44Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q06253:p.Tyr47Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q06253:p.Lys48Met 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 976555 unspecified role unspecified role bait bait direct interaction direct interaction false false false Fig 3A, S3A. Wild-type Phd binding to OR1/2 (operator region of the phd/doc operon containing the two binding sites). The affinity of these mutants for OR1/2 is much weaker than that of the wild-type protein. phd_doc_ta-1 976549 n/a 976553 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 976555 unspecified role unspecified role bait bait n/a 976554 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) physical association physical association false false false Fig 3B,3F, S3C, 5A. Compared to wild-type Phd, the mutants PhdF44A and PhdY47A show little if any increase in their affinity for the OR1/2 DNA in presence of Doc. Phd mutants F44A and Y47A bind to Doc, but this does not lead to an increase of the affinity of the Phd mutant for OR1. Mutant K48M shows weak but observable enhancement of Phd2 binding to OR1 in the presence of Doc. Doc mutants H66Y/N78W, and H66Y/A61R show a less complex set of bands in EMSA. phd_doc-1 976550 n/a 976553 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a 976554 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) Stoichiometry: 3.0 direct interaction direct interaction false false false Fig 4, Fig S4, Table 1. working 25.7%, free 28.5% 2.40 A The asymmetric unit of the Phd/Doc cocrystals contain three copies of Phd and one copy of Doc, which through crystal symmetry form a hetero-octameric Phd2-Doc-Phd2-Doc-Phd2 complex. Doc Site H (encompassing residues from alpha helices a1, a4, a5, and the loop a4-a5 of Doc) binds to Phd segment Phe56-Arg73. Doc Site L (mainly encompassing residues from loops a1-a2 (residues 28�31) and a5-a6 (residues106�111) and from helices a2 (34�35, 38�39, 42) and a3 (57�61, 64�65)), binds to Leu52-Thr62 of Phd. phd_phd 976550 n/a 976554 stimulator stimulator neutral component neutral component n terminal 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: 1.0 n/a 976554 unspecified role unspecified role neutral component neutral component n terminal 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: 1.0 direct interaction direct interaction false false false Fig 1A. working 23%, free 27.1% 2.20 A This N-terminal domain forms a small globular homodimer comprising a central six-stranded beta sheet decorated with four alpha helices (two from each monomer), resembling the antitoxin YefM (Kamada and Hanaoka, 2005). phd_ta_dna 976551 n/a 976555 unspecified role unspecified role neutral component neutral component n/a 976554 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Fig 2A. An alpha helix is induced in Phd upon addition of operator DNA in vitro. phd_doc 976552 n/a 976553 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a 976554 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false Fig 2C, Table S4. The best model that explains the binding data involves two Phd dimers binding to a Doc monomer with affinities of 0.35 uM and 31 uM. 25 degrees C in 20 mM HEPES pH 7.5.