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/ The SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster transfer. The SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster transfer. Biochemistry (0006-2960) 2009 imex curation Chahal H.K., Dai Y., Saini A., Ayala-Castro C., Outten F.W. SufA was crosslinked with Mts-Atf-biotin {2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-l-lysinyl]ethylmethane thiosulfonate} (Pierce). The Western blot detected the biotin moiety of the crosslinker with streptavidin-conjugated HRP. Complexes were purified by gel filtration. wayne.outten@chem.sc.edu 13-Jan-2014: Contacted by IntAct-Help. Only protein-protein interactions 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 anti tag western anti tag western blot anti tag western Chahal H.K., Dai Y., Saini A., Ayala-Castro C., Outten F.W. SufA was crosslinked with Mts-Atf-biotin {2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-l-lysinyl]ethylmethane thiosulfonate} (Pierce). The Western blot detected the biotin moiety of the crosslinker with streptavidin-conjugated HRP. Complexes were purified by gel filtration. wayne.outten@chem.sc.edu Accepted 2013-DEC-02 by PPORRAS Biochemistry (0006-2960) 2009 Only protein-protein interactions imex curation The SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster transfer. The SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster transfer. Biochemistry (0006-2960) 2009 imex curation Chahal H.K., Dai Y., Saini A., Ayala-Castro C., Outten F.W. SufA was crosslinked with Mts-Atf-biotin {2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-l-lysinyl]ethylmethane thiosulfonate} (Pierce). The Western blot detected the biotin moiety of the crosslinker with streptavidin-conjugated HRP. Complexes were purified by gel filtration. wayne.outten@chem.sc.edu 13-Jan-2014: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Chahal H.K., Dai Y., Saini A., Ayala-Castro C., Outten F.W. SufA was crosslinked with Mts-Atf-biotin {2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-l-lysinyl]ethylmethane thiosulfonate} (Pierce). The Western blot detected the biotin moiety of the crosslinker with streptavidin-conjugated HRP. Complexes were purified by gel filtration. wayne.outten@chem.sc.edu Accepted 2013-DEC-02 by PPORRAS Biochemistry (0006-2960) 2009 Only protein-protein interactions imex curation The SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster transfer. The SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster transfer. Biochemistry (0006-2960) 2009 imex curation Chahal H.K., Dai Y., Saini A., Ayala-Castro C., Outten F.W. SufA was crosslinked with Mts-Atf-biotin {2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-l-lysinyl]ethylmethane thiosulfonate} (Pierce). The Western blot detected the biotin moiety of the crosslinker with streptavidin-conjugated HRP. Complexes were purified by gel filtration. wayne.outten@chem.sc.edu 13-Jan-2014: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro cd circular dichroism CD CD cd predetermined predetermined participant predetermined Chahal H.K., Dai Y., Saini A., Ayala-Castro C., Outten F.W. Changes in CD spectra indicate Fe-S cluster transfer from holoSufBCD to apoSufA. SufA was crosslinked with Mts-Atf-biotin {2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-l-lysinyl]ethylmethane thiosulfonate} (Pierce). The Western blot detected the biotin moiety of the crosslinker with streptavidin-conjugated HRP. Complexes were purified by gel filtration. wayne.outten@chem.sc.edu Accepted 2013-DEC-02 by PPORRAS Biochemistry (0006-2960) 2009 Only protein-protein interactions imex curation The SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster transfer. The SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster transfer. Biochemistry (0006-2960) 2009 imex curation Chahal H.K., Dai Y., Saini A., Ayala-Castro C., Outten F.W. SufA was crosslinked with Mts-Atf-biotin {2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-l-lysinyl]ethylmethane thiosulfonate} (Pierce). The Western blot detected the biotin moiety of the crosslinker with streptavidin-conjugated HRP. Complexes were purified by gel filtration. wayne.outten@chem.sc.edu 13-Jan-2014: Contacted by IntAct-Help. 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 Chahal H.K., Dai Y., Saini A., Ayala-Castro C., Outten F.W. Changes in the UV-visible absorption spectra indicate Fe-S cluster transfer from a holo-protein to an apo-protein/complex. SufA was crosslinked with Mts-Atf-biotin {2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-l-lysinyl]ethylmethane thiosulfonate} (Pierce). The Western blot detected the biotin moiety of the crosslinker with streptavidin-conjugated HRP. Complexes were purified by gel filtration. wayne.outten@chem.sc.edu Accepted 2013-DEC-02 by PPORRAS Biochemistry (0006-2960) 2009 Only protein-protein interactions imex curation The SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster transfer. The SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster transfer. Biochemistry (0006-2960) 2009 imex curation Chahal H.K., Dai Y., Saini A., Ayala-Castro C., Outten F.W. SufA was crosslinked with Mts-Atf-biotin {2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-l-lysinyl]ethylmethane thiosulfonate} (Pierce). The Western blot detected the biotin moiety of the crosslinker with streptavidin-conjugated HRP. Complexes were purified by gel filtration. wayne.outten@chem.sc.edu 13-Jan-2014: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro pull down pull down weight by comassie molecular weight estimation by coomasie staining weight by comassie Chahal H.K., Dai Y., Saini A., Ayala-Castro C., Outten F.W. SufA was crosslinked with Mts-Atf-biotin {2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-l-lysinyl]ethylmethane thiosulfonate} (Pierce). The Western blot detected the biotin moiety of the crosslinker with streptavidin-conjugated HRP. Complexes were purified by gel filtration. wayne.outten@chem.sc.edu Accepted 2013-DEC-02 by PPORRAS Biochemistry (0006-2960) 2009 Only protein-protein interactions imex curation fe-s cluster iron-sulfur cluster small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) fas ferrous ammonium sulfate small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) sufa_ecoli Protein SufA sufA b1684 JW1674 ydiC protein protein ecoli Escherichia coli (strain K12) MDMHSGTFNPQDFAWQGLTLTPAAAIHIRELVAKQPGMVGVRLGVKQTGCAGFGYVLDSVSEPDKDDLLFEHDGAKLFVPLQAMPFIDGTEVDFVREGLNQIFKFHNPKAQNECGCGESFGV D26817F42E4B740B sufc_ecoli Probable ATP-dependent transporter SufC ynhD sufC b1682 JW1672 protein protein ecoli Escherichia coli (strain K12) MLSIKDLHVSVEDKAILRGLSLDVHPGEVHAIMGPNGSGKSTLSATLAGREDYEVTGGTVEFKGKDLLALSPEDRAGEGIFMAFQYPVEIPGVSNQFFLQTALNAVRSYRGQETLDRFDFQDLMEEKIALLKMPEDLLTRSVNVGFSGGEKKRNDILQMAVLEPELCILDESDSGLDIDALKVVADGVNSLRDGKRSFIIVTHYQRILDYIKPDYVHVLYQGRIVKSGDFTLVKQLEEQGYGWLTEQQ DIP protein P77499 original sequence version: null 78FB1108E1850DF9 sufs_ecoli Cysteine desulfurase sufS csdB b1680 JW1670 ynhB Selenocysteine lyase Selenocysteine reductase Selenocysteine beta-lyase protein protein ecoli Escherichia coli (strain K12) MIFSVDKVRADFPVLSREVNGLPLAYLDSAASAQKPSQVIDAEAEFYRHGYAAVHRGIHTLSAQATEKMENVRKRASLFINARSAEELVFVRGTTEGINLVANSWGNSNVRAGDNIIISQMEHHANIVPWQMLCARVGAELRVIPLNPDGTLQLETLPTLFDEKTRLLAITHVSNVLGTENPLAEMITLAHQHGAKVLVDGAQAVMHHPVDVQALDCDFYVFSGHKLYGPTGIGILYVKEALLQEMPPWEGGGSMIATVSLSEGTTWTKAPWRFEAGTPNTGGIIGLGAALEYVSALGLNNIAEYEQNLMHYALSQLESVPDLTLYGPQNRLGVIAFNLGKHHAYDVGSFLDNYGIAVRTGHHCAMPLMAYYNVPAMCRASLAMYNTHEEVDRLVTGLQRIHRLLG 9374C43C3AD9D8E3 sufe_ecoli Cysteine desulfuration protein SufE sufE b1679 JW1669 ynhA protein protein ecoli Escherichia coli (strain K12) MALLPDKEKLLRNFLRCANWEEKYLYIIELGQRLPELRDEDRSPQNSIQGCQSQVWIVMRQNAQGIIELQGDSDAAIVKGLIAVVFILYDQMTPQDIVNFDVRPWFEKMALTQHLTPSRSQGLEAMIRAIRAKAAALS DIP protein P76194 original sequence version: 72 AD7B89A74C946772 sufd_ecoli FeS cluster assembly protein SufD ynhC sufD b1681 JW1671 protein protein ecoli Escherichia coli (strain K12) MAGLPNSSNALQQWHHLFEAEGTKRSPQAQQHLQQLLRTGLPTRKHENWKYTPLEGLINSQFVSIAGEISPQQRDALALTLDSVRLVFVDGRYVPALSDATEGSGYEVSINDDRQGLPDAIQAEVFLHLTESLAQSVTHIAVKRGQRPAKPLLLMHITQGVAGEEVNTAHYRHHLDLAEGAEATVIEHFVSLNDARHFTGARFTINVAANAHLQHIKLAFENPLSHHFAHNDLLLAEDATAFSHSFLLGGAVLRHNTSTQLNGENSTLRINSLAMPVKNEVCDTRTWLEHNKGFCNSRQLHKTIVSDKGRAVFNGLINVAQHAIKTDGQMTNNNLLMGKLAEVDTKPQLEIYADDVKCSHGATVGRIDDEQIFYLRSRGINQQDAQQMIIYAFAAELTEALRDEGLKQQVLARIGQRLPGGAR DIP protein P77689 original sequence version: null 5744291F1E0AE825 sodium sulfide sodium sulfide small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) sufb_ecoli FeS cluster assembly protein SufB ynhE sufB b1683 JW5273 protein protein ecoli Escherichia coli (strain K12) MSRNTEATDDVKTWTGGPLNYKEGFFTQLATDELAKGINEEVVRAISAKRNEPEWMLEFRLNAYRAWLEMEEPHWLKAHYDKLNYQDYSYYSAPSCGNCDDTCASEPGAVQQTGANAFLSKEVEAAFEQLGVPVREGKEVAVDAIFDSVSVATTYREKLAEQGIIFCSFGEAIHDHPELVRKYLGTVVPGNDNFFAALNAAVASDGTFIYVPKGVRCPMELSTYFRINAEKTGQFERTILVADEDSYVSYIEGCSAPVRDSYQLHAAVVEVIIHKNAEVKYSTVQNWFPGDNNTGGILNFVTKRALCEGENSKMSWTQSETGSAITWKYPSCILRGDNSIGEFYSVALTSGHQQADTGTKMIHIGKNTKSTIISKGISAGHSQNSYRGLVKIMPTATNARNFTQCDSMLIGANCGAHTFPYVECRNNSAQLEHEATTSRIGEDQLFYCLQRGISEEDAISMIVNGFCKDVFSELPLEFAVEAQKLLAISLEHSVG DIP protein P77522 original sequence version: null 3BB49EEADB6C1278 l-cysteine L-cysteine (2R)-2-amino-3-mercaptopropanoic acid (2R)-2-amino-3-sulfanylpropanoic acid (R)-2-amino-3-mercaptopropanoic acid C Cys CYSTEINE E920 FREE CYSTEINE L-2-Amino-3-mercaptopropionic acid L-Cystein L-Cysteine L-Zystein L-cysteine small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) XUJNEKJLAYXESH-REOHCLBHSA-N XUJNEKJLAYXESH-REOHCLBHSA-N sufa-sufbcd-1 1 14 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 12 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association Fig. 1b, 2, S1a The label transfer is more efficient if SufA binds to the SufBCD complex than if it binds to SufB (EBI-8805150) or SufC (EBI-8805163) alone. SufE (P76194) and SufS (P77444) on their own or in concert slightly inhibit the label transfer from SufA to SufBCD complex (Fig. 2). The inhibition is relatively proportional to SufE or SufS concentration. sufa-sufb-1 1 14 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 1 The label transfer is more efficient if SufA binds to the SufBCD complex (EBI-8805115) than if it binds to SufB alone. sufa-sufc-1 1 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 1a, S1a Overall label transfer is more efficient if SufA binds to the SufBCD complex (EBI-8805115) than if it binds to SufC alone. However, label transfer from SufA directly to SufC is more efficient if only SufA and SufC are in the reaction. sufa-sufs 2 10 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) direct interaction direct interaction SufE (P76194) Fig. S2a sufa-sufbcd 4 7 ancillary ancillary neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 15 ancillary ancillary neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 11 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) 10 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) 12 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 14 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association Figs 3 sufa-sufbcd-3 3 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 14 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 12 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association Fig. 6 SufBCD was reconstituted with a Fe-S cluster by preincubating it with SufSE/L-cysteine and FAS and purification with anion exchange chromatography prior to the addition of SufA. sufa-fas 3 13 unspecified role unspecified role neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 7 unspecified role unspecified role neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association Fig. 7 EDTA disrupts the transfer of the Fe-S cluster to SufA. fas-sufbcd 4 11 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) 15 ancillary ancillary neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 10 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 12 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 14 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 7 ancillary ancillary neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association Fig. S2b sufa-sufbcd-5 5 8 unspecified role unspecified role bait bait 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 ecoli Escherichia coli (strain K12) 14 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 12 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) physical association physical association Fig. 8 The interaction was enhanced if SufBCD was bound to the Fe-S cluster (holoprotein form). sufa-fe_s 3 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) direct interaction direct interaction Fig. S3b EDTA inhibits holoprotein association in a concentration-dependent manner. sufbcd-fe_s 3 6 unspecified role unspecified role neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 12 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 14 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association Fig. S3b EDTA inhibits holoprotein association in a concentration-dependent manner. sufbcd-fas-l-cys 4 7 ancillary ancillary neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 14 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 11 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) 10 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) 15 ancillary ancillary neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association Figs 3b sufa-fas-l-cys 4 15 ancillary ancillary neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 10 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) 11 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) 7 ancillary ancillary neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association Figs 3a sufbcd-sufa-1 4 9 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 14 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 12 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association Figs 4 SufBCD complex was preincubated with SufSE/L-cysteine and FAS to reconstitute the Fe-S cluster on SufBCD. The complex was then purified by anion exchange chromatography before adding SufA. (The opposite direction of reconstituting Fe-S cluster on SufA and then adding SufBCD resulted in no interactions, see Fig 5).