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/ layer-2007-1 SufE transfers sulfur from SufS to SufB for iron-sulfur cluster assembly. in vitro In vitro bifunctional agent crosslink protein cross-linking with a bifunctional reagent Label transfer techniques Photoaffinity labelling Label transfer techniques Photoaffinity labelling bifunctional agent crosslink tag visualisation tag visualisation Layer G., Gaddam S.A., Ayala-Castro C.N., Ollagnier-de Choudens S., Lascoux D., Fontecave M., Outten F.W. SufE was crosslinked with trifunctional cross-linker Mts-Atf-biotin (2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-l-lysinyl]ethyl methane-thiosulfonate (Pierce) and purified by gel filtration. Mts group; (Sulfhydryl-reactive methanethiosulfonate) – labels a purified "bait" protein at the side chain of reduced cysteine residues. Atf group; UV light-activatable perfluorophenylazide – for crosslinking nonspecifically to interacting (prey) proteins. Biotin group – remains attached to target interacting protein after disulfide bond cleavage, thereby tagging the previously unknown interacting protein(s). After photoactivation of the Atf moiety, the interacting complex is isolated and the disulfide bond reduced, resulting in the biotin label being transferred to the interacting protein. Proteins were separated by SDS-PAGE and Biotin-bound interacting proteins detected with streptavidin-conjugated HRP. sollagnier@cea.fr,wayne.outten@chem.sc.edu J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation layer-2007-2 SufE transfers sulfur from SufS to SufB for iron-sulfur cluster assembly. in vitro In vitro pull down pull down weight by comassie molecular weight estimation by coomasie staining weight by comassie Layer G., Gaddam S.A., Ayala-Castro C.N., Ollagnier-de Choudens S., Lascoux D., Fontecave M., Outten F.W. SufE was crosslinked with trifunctional cross-linker Mts-Atf-biotin (2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-l-lysinyl]ethyl methane-thiosulfonate (Pierce) and purified by gel filtration. Mts group; (Sulfhydryl-reactive methanethiosulfonate) – labels a purified "bait" protein at the side chain of reduced cysteine residues. Atf group; UV light-activatable perfluorophenylazide – for crosslinking nonspecifically to interacting (prey) proteins. Biotin group – remains attached to target interacting protein after disulfide bond cleavage, thereby tagging the previously unknown interacting protein(s). After photoactivation of the Atf moiety, the interacting complex is isolated and the disulfide bond reduced, resulting in the biotin label being transferred to the interacting protein. Proteins were separated by SDS-PAGE and Biotin-bound interacting proteins detected with streptavidin-conjugated HRP. sollagnier@cea.fr,wayne.outten@chem.sc.edu J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation layer-2007-3 SufE transfers sulfur from SufS to SufB for iron-sulfur cluster assembly. in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Layer G., Gaddam S.A., Ayala-Castro C.N., Ollagnier-de Choudens S., Lascoux D., Fontecave M., Outten F.W. SufE was crosslinked with trifunctional cross-linker Mts-Atf-biotin (2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-l-lysinyl]ethyl methane-thiosulfonate (Pierce) and purified by gel filtration. Mts group; (Sulfhydryl-reactive methanethiosulfonate) – labels a purified "bait" protein at the side chain of reduced cysteine residues. Atf group; UV light-activatable perfluorophenylazide – for crosslinking nonspecifically to interacting (prey) proteins. Biotin group – remains attached to target interacting protein after disulfide bond cleavage, thereby tagging the previously unknown interacting protein(s). After photoactivation of the Atf moiety, the interacting complex is isolated and the disulfide bond reduced, resulting in the biotin label being transferred to the interacting protein. Proteins were separated by SDS-PAGE and Biotin-bound interacting proteins detected with streptavidin-conjugated HRP. sollagnier@cea.fr,wayne.outten@chem.sc.edu J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation layer-2007-4 SufE transfers sulfur from SufS to SufB for iron-sulfur cluster assembly. in vitro In vitro enzymatic study enzymatic study ms participant Identification by mass spectrometry ms participant Layer G., Gaddam S.A., Ayala-Castro C.N., Ollagnier-de Choudens S., Lascoux D., Fontecave M., Outten F.W. SufE was crosslinked with trifunctional cross-linker Mts-Atf-biotin (2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-l-lysinyl]ethyl methane-thiosulfonate (Pierce) and purified by gel filtration. Mts group; (Sulfhydryl-reactive methanethiosulfonate) – labels a purified "bait" protein at the side chain of reduced cysteine residues. Atf group; UV light-activatable perfluorophenylazide – for crosslinking nonspecifically to interacting (prey) proteins. Biotin group – remains attached to target interacting protein after disulfide bond cleavage, thereby tagging the previously unknown interacting protein(s). After photoactivation of the Atf moiety, the interacting complex is isolated and the disulfide bond reduced, resulting in the biotin label being transferred to the interacting protein. Proteins were separated by SDS-PAGE and Biotin-bound interacting proteins detected with streptavidin-conjugated HRP. sollagnier@cea.fr,wayne.outten@chem.sc.edu J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation layer-2007-5 SufE transfers sulfur from SufS to SufB for iron-sulfur cluster assembly. in vitro In vitro enzymatic study enzymatic study predetermined predetermined participant predetermined Layer G., Gaddam S.A., Ayala-Castro C.N., Ollagnier-de Choudens S., Lascoux D., Fontecave M., Outten F.W. SufE was crosslinked with trifunctional cross-linker Mts-Atf-biotin (2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-l-lysinyl]ethyl methane-thiosulfonate (Pierce) and purified by gel filtration. Mts group; (Sulfhydryl-reactive methanethiosulfonate) – labels a purified "bait" protein at the side chain of reduced cysteine residues. Atf group; UV light-activatable perfluorophenylazide – for crosslinking nonspecifically to interacting (prey) proteins. Biotin group – remains attached to target interacting protein after disulfide bond cleavage, thereby tagging the previously unknown interacting protein(s). After photoactivation of the Atf moiety, the interacting complex is isolated and the disulfide bond reduced, resulting in the biotin label being transferred to the interacting protein. Proteins were separated by SDS-PAGE and Biotin-bound interacting proteins detected with streptavidin-conjugated HRP. sollagnier@cea.fr,wayne.outten@chem.sc.edu J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation sufs_ecoli Cysteine desulfurase sufS csdB b1680 JW1670 ynhB Selenocysteine lyase Selenocysteine reductase Selenocysteine beta-lyase protein protein ecoli Escherichia coli (strain K12) MIFSVDKVRADFPVLSREVNGLPLAYLDSAASAQKPSQVIDAEAEFYRHGYAAVHRGIHTLSAQATEKMENVRKRASLFINARSAEELVFVRGTTEGINLVANSWGNSNVRAGDNIIISQMEHHANIVPWQMLCARVGAELRVIPLNPDGTLQLETLPTLFDEKTRLLAITHVSNVLGTENPLAEMITLAHQHGAKVLVDGAQAVMHHPVDVQALDCDFYVFSGHKLYGPTGIGILYVKEALLQEMPPWEGGGSMIATVSLSEGTTWTKAPWRFEAGTPNTGGIIGLGAALEYVSALGLNNIAEYEQNLMHYALSQLESVPDLTLYGPQNRLGVIAFNLGKHHAYDVGSFLDNYGIAVRTGHHCAMPLMAYYNVPAMCRASLAMYNTHEEVDRLVTGLQRIHRLLG sufe_ecoli Cysteine desulfuration protein SufE sufE b1679 JW1669 ynhA protein protein ecoli Escherichia coli (strain K12) MALLPDKEKLLRNFLRCANWEEKYLYIIELGQRLPELRDEDRSPQNSIQGCQSQVWIVMRQNAQGIIELQGDSDAAIVKGLIAVVFILYDQMTPQDIVNFDVRPWFEKMALTQHLTPSRSQGLEAMIRAIRAKAAALS sufc_ecoli Probable ATP-dependent transporter SufC ynhD sufC b1682 JW1672 protein protein ecoli Escherichia coli (strain K12) MLSIKDLHVSVEDKAILRGLSLDVHPGEVHAIMGPNGSGKSTLSATLAGREDYEVTGGTVEFKGKDLLALSPEDRAGEGIFMAFQYPVEIPGVSNQFFLQTALNAVRSYRGQETLDRFDFQDLMEEKIALLKMPEDLLTRSVNVGFSGGEKKRNDILQMAVLEPELCILDESDSGLDIDALKVVADGVNSLRDGKRSFIIVTHYQRILDYIKPDYVHVLYQGRIVKSGDFTLVKQLEEQGYGWLTEQQ sufb_ecoli FeS cluster assembly protein SufB ynhE sufB b1683 JW5273 protein protein ecoli Escherichia coli (strain K12) MSRNTEATDDVKTWTGGPLNYKEGFFTQLATDELAKGINEEVVRAISAKRNEPEWMLEFRLNAYRAWLEMEEPHWLKAHYDKLNYQDYSYYSAPSCGNCDDTCASEPGAVQQTGANAFLSKEVEAAFEQLGVPVREGKEVAVDAIFDSVSVATTYREKLAEQGIIFCSFGEAIHDHPELVRKYLGTVVPGNDNFFAALNAAVASDGTFIYVPKGVRCPMELSTYFRINAEKTGQFERTILVADEDSYVSYIEGCSAPVRDSYQLHAAVVEVIIHKNAEVKYSTVQNWFPGDNNTGGILNFVTKRALCEGENSKMSWTQSETGSAITWKYPSCILRGDNSIGEFYSVALTSGHQQADTGTKMIHIGKNTKSTIISKGISAGHSQNSYRGLVKIMPTATNARNFTQCDSMLIGANCGAHTFPYVECRNNSAQLEHEATTSRIGEDQLFYCLQRGISEEDAISMIVNGFCKDVFSELPLEFAVEAQKLLAISLEHSVG sufd_ecoli FeS cluster assembly protein SufD ynhC sufD b1681 JW1671 protein protein ecoli Escherichia coli (strain K12) MAGLPNSSNALQQWHHLFEAEGTKRSPQAQQHLQQLLRTGLPTRKHENWKYTPLEGLINSQFVSIAGEISPQQRDALALTLDSVRLVFVDGRYVPALSDATEGSGYEVSINDDRQGLPDAIQAEVFLHLTESLAQSVTHIAVKRGQRPAKPLLLMHITQGVAGEEVNTAHYRHHLDLAEGAEATVIEHFVSLNDARHFTGARFTINVAANAHLQHIKLAFENPLSHHFAHNDLLLAEDATAFSHSFLLGGAVLRHNTSTQLNGENSTLRINSLAMPVKNEVCDTRTWLEHNKGFCNSRQLHKTIVSDKGRAVFNGLINVAQHAIKTDGQMTNNNLLMGKLAEVDTKPQLEIYADDVKCSHGATVGRIDDEQIFYLRSRGINQQDAQQMIIYAFAAELTEALRDEGLKQQVLARIGQRLPGGAR sufe-sufs-1 1455481 n/a 1455486 unspecified role unspecified role prey prey n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) n/a 1455487 unspecified role unspecified role bait bait mts-atf tag on cysteines tag tag certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false cys17/cys51 biotin tag biotin tag certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Fig. 1a, 1b (lane 2) In Fig. 1b, lane 2, SufA has been added to SufE-SufS; here, the SufA band is not visible but appears to reduce the label-transfer activity compared to the interaction in Fig. 1a. sufe-sufabcds-1 1455481 n/a 1455488 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1455489 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1455487 unspecified role unspecified role bait bait mts-atf tag on cysteines tag tag certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false cys17/cys51 biotin tag biotin tag certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1455486 unspecified role unspecified role prey prey n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) physical association physical association false false false Fig. 1b (lanes 3 & 5) The SufA (lane 5 only) and SufD bands are not visible, SufE and SufC bands are weak. When SufA is added (lane 5) all bands become weak. sufe-sufabcd-1 1455481 n/a 1455489 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1455490 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1455487 unspecified role unspecified role bait bait mts-atf tag on cysteines tag tag certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false cys17/cys51 biotin tag biotin tag certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1455488 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) physical association physical association false false false Fig. 1b (lanes 1 & 4) The SufE, SufC and SufD bands are weak and the SufA band is not visible (lane 4 only). When SufA is added, the SufB band becomes weaker (lane 4). sufe-sufbcd-2 1455482 n/a 1455487 unspecified role unspecified role bait bait c-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term 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 ecoli Escherichia coli (strain K12) n/a 1455490 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1455489 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1455488 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) physical association physical association false false false Fig. 2a, b, S2a, S2b This interaction is true under aerobic conditions, anaerobic conditions and after threatment with iodacetamide (blocks free cysteine residues). sufe-sufbc-2 1455482 n/a 1455487 unspecified role unspecified role bait bait c-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term 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 ecoli Escherichia coli (strain K12) n/a 1455488 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1455489 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) physical association physical association false false false Fig. 2b It is not clear from the publication whether this interactions was tested under aerobic or anaerobic conditions - or both. sufe-sufs-3 1455483 n/a 1455486 unspecified role unspecified role prey prey n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) n/a 1455487 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false 1455483 Fig. 3a, S3a sufe-sufbcd-3 1455483 n/a 1455490 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1455487 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) n/a 1455488 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1455489 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) physical association physical association false false false 1455483 Fig. 3b, S3b sufse-sufbcd-1 1455484 n/a 1455490 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1455488 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1455489 enzyme target enzyme target substrate substrate neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1455487 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1455486 enzyme enzyme neutral component neutral component n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false Fig. 4b SufS transfers up to 7 sulfur atoms to SufB as shown by the MS profile of SufB. MS profiles for SufC and SufD did not vary when SufS and cysteine were added to the reaction. sufe-sufbcd-4 1455484 n/a 1455489 enzyme target enzyme target substrate substrate neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1455487 enzyme enzyme neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1455488 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1455490 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false Fig. S5b sufse-sufbc-1 1455485 n/a 1455487 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1455488 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1455489 enzyme target enzyme target substrate substrate neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1455486 enzyme enzyme neutral component neutral component n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false Fig. S4 sufse-sufbcd-2 1455485 n/a 1455486 enzyme enzyme neutral component neutral component n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) n/a 1455490 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1455488 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1455487 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1455489 enzyme target enzyme target substrate substrate neutral component neutral component ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false Fig. S4