MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Thioredoxin-like protein TlpA from Bradyrhizobium japonicum is a reductant for the copper metallochaperone ScoI. Thioredoxin-like protein TlpA from Bradyrhizobium japonicum is a reductant for the copper metallochaperone ScoI. FEBS Lett. (0014-5793) 2012 Mohorko E., Abicht HK., Bühler D., Glockshuber R., Hennecke H., Fischer HM. in vitro In vitro crosslink cross-linking study crosslink weight by comassie molecular weight estimation by coomasie staining weight by comassie Mohorko E., Abicht HK., Bühler D., Glockshuber R., Hennecke H., Fischer HM. FEBS Lett. (0014-5793) 2012 Thioredoxin-like protein TlpA from Bradyrhizobium japonicum is a reductant for the copper metallochaperone ScoI. Thioredoxin-like protein TlpA from Bradyrhizobium japonicum is a reductant for the copper metallochaperone ScoI. FEBS Lett. (0014-5793) 2012 Mohorko E., Abicht HK., Bühler D., Glockshuber R., Hennecke H., Fischer HM. in vitro In vitro oxidoreduct assay oxidoreductase assay oxidoreduct assay predetermined predetermined participant predetermined Mohorko E., Abicht HK., Bühler D., Glockshuber R., Hennecke H., Fischer HM. FEBS Lett. (0014-5793) 2012 q89vb6_bradu blr1131 protein protein braja Bradyrhizobium japonicum (strain USDA 110) MSSATRPLVIATAFAASLIVGLLIMFWAMGGVGKVAQPAAIGGPFQLTDQNGKAVTDKSLKGKPTLIFFGYTHCPDVCPTSLFEISEVLRAMGKDADKVNAIFISVDPERDTPATMKNYLSSFDPHLEGLSGDPAEIAKVITSYRVYAKKVPTKDGDYTMDHTALIYLMDRDGRFVSPFNLKRTPEEAAADLKRYL mint 191812EA51D55073 tlpa_bradu Thiol:disulfide interchange protein TlpA tlpA Cytochrome c biogenesis protein TlpA bll1380 protein protein braja Bradyrhizobium japonicum (strain USDA 110) MLDTKPSATRRIPLVIATVAVGGLAGFAALYGLGLSRAPTGDPACRAAVATAQKIAPLAHGEVAALTMASAPLKLPDLAFEDADGKPKKLSDFRGKTLLVNLWATWCVPCRKEMPALDELQGKLSGPNFEVVAINIDTRDPEKPKTFLKEANLTRLGYFNDQKAKVFQDLKAIGRALGMPTSVLVDPQGCEIATIAGPAEWASEDALKLIRAATGKAAAAL mint DD4C7EADF2F0FE1B q89vb6-tlpa 1 ScoI 3 weight by comassie molecular weight estimation by coomasie staining weight by comassie experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified Q89VB6:p.Cys78Ser mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain C S Tue Jul 12 10:23:00 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 18:46:37 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'cys78ser' Tue Jun 30 17:02:56 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys78Ser' TlpA 4 weight by comassie molecular weight estimation by coomasie staining weight by comassie experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction f3 mint we used TlpA-C110S and either wild-type ScoI, or ScoI-C74S, or ScoI-C78S as the interacting partners. Soluble TlpA-C110S was activated with DTNB, incubated with soluble, reduced ScoI, and iodoacetamide was finally added to quench free thiols. Reaction products were analyzed with non-reducing and reducing SDS-PAGE. The occurrence of a band at 38 kDa, reflecting the sum of the molecular masses of TlpA and ScoI, indicated that a mixed disulfide had formed (Fig. 3). This band was not seen in reducing SDS-PAGE The 38-kDa heterodisulfide from the reaction between TlpA-C110S and ScoI-C74S was subjected to tryptic digestion followed by MALDI-MS (data not shown). A large tryptic peptide with the mass of 5,146.8 Da was detected which can only be explained as being the disulfide-bridged product of tryptic peptides from the active sites (underscored) of TlpA-C110S (97TLLVNLWATWCVPSRK112; 1,888.3 Da) and ScoI-C74S (62GKPTLIFFGYTHSPDVCPTSLFEISEVLR90; 3,255.8 Da). Little heterodisulfide formed at a slow rate between TlpA-C110S and ScoI-C78S. By contrast, the TlpA-C110SScoI-C74S heterodisulfide formed rapidly and stayed remarkably stable (Fig. 3), suggesting that C78 is indeed the preferred target, as was rationalized before. The disulfide between TlpA-C110S and wildtype ScoI was apparently resolved over time by the still intact C74. Homodimers of the individual proteins did not form under these conditions. q89vb6-tlpa-1 1 TlpA 4 weight by comassie molecular weight estimation by coomasie staining weight by comassie experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified ScoI 3 weight by comassie molecular weight estimation by coomasie staining weight by comassie experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified Q89VB6:p.Cys74Ser mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain C S Tue Jul 12 10:21:43 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 17:16:45 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'cys74ser' Tue Jun 30 16:03:26 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys74Ser' direct interaction direct interaction f3 mint we used TlpA-C110S and either wild-type ScoI, or ScoI-C74S, or ScoI-C78S as the interacting partners. Soluble TlpA-C110S was activated with DTNB, incubated with soluble, reduced ScoI, and iodoacetamide was finally added to quench free thiols. Reaction products were analyzed with non-reducing and reducing SDS-PAGE. The occurrence of a band at 38 kDa, reflecting the sum of the molecular masses of TlpA and ScoI, indicated that a mixed disulfide had formed (Fig. 3). This band was not seen in reducing SDS-PAGE The 38-kDa heterodisulfide from the reaction between TlpA-C110S and ScoI-C74S was subjected to tryptic digestion followed by MALDI-MS (data not shown). A large tryptic peptide with the mass of 5,146.8 Da was detected which can only be explained as being the disulfide-bridged product of tryptic peptides from the active sites (underscored) of TlpA-C110S (97TLLVNLWATWCVPSRK112; 1,888.3 Da) and ScoI-C74S (62GKPTLIFFGYTHSPDVCPTSLFEISEVLR90; 3,255.8 Da). Little heterodisulfide formed at a slow rate between TlpA-C110S and ScoI-C78S. By contrast, the TlpA-C110SScoI-C74S heterodisulfide formed rapidly and stayed remarkably stable (Fig. 3), suggesting that C78 is indeed the preferred target, as was rationalized before. The disulfide between TlpA-C110S and wildtype ScoI was apparently resolved over time by the still intact C74. Homodimers of the individual proteins did not form under these conditions. q89vb6-tlpa-2 2 ScoI 3 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp electron acceptor electron acceptor neutral component neutral component purified purified TlpA 4 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp electron donor electron donor neutral component neutral component purified purified redox reaction oxidoreductase activity electron transfer reaction redox reaction f2b mint Figure 2b shows the fluorescence trace of a reaction initiated by mixing equimolar amounts of ScoIox and TlpAred (final initial concentrations 2 M). Using the known ratio between the forward (k2) and reverse (k2) reaction (Keq= 1,760 = k2/k2), we obtained rate constants of 9.4 x 104 M1 s1 and 53 M1 s1 for k2 and k2, respectively. Hence, the rate of ScoIox reduction by TlpAred was in a range typically observed for physiologically relevant disulfide exchange reactions, which provided a strong hint that TlpA is a reductant of ScoI in vivo.