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/ kulp-2006-2 Domain architecture of protein-disulfide isomerase facilitates its dual role as an oxidase and an isomerase in Ero1p-mediated disulfide formation. in vitro In vitro fluorescence spectr classical fluorescence spectroscopy fluorescence spectr predetermined predetermined participant predetermined Kulp MS., Frickel EM., Ellgaard L., Weissman JS. BioCreative - Critical Assessment of Information Extraction systems in Biology The rate at which the enzymes catalysed the formation of the correct disulfide bonds in RNase A was monitored by following RNase activity (hydrolysis of cCMP) in the presence of Ero1p. weissman@cmp.ucsf.edu J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation kulp-2006-1 Domain architecture of protein-disulfide isomerase facilitates its dual role as an oxidase and an isomerase in Ero1p-mediated disulfide formation. in vitro In vitro enzymatic study enzymatic study predetermined predetermined participant predetermined Kulp MS., Frickel EM., Ellgaard L., Weissman JS. BioCreative - Critical Assessment of Information Extraction systems in Biology The rate at which the enzymes catalysed the formation of the correct disulfide bonds in RNase A was monitored by following RNase activity (hydrolysis of cCMP) in the presence of Ero1p. weissman@cmp.ucsf.edu J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation rnas1_bovin Ribonuclease pancreatic RNASE1 RNS1 RNase 1 RNase A protein protein bovin Bos taurus (Bovine) Bovine MALKSLVLLSLLVLVLLLVRVQPSLGKETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV pdi_yeast Protein disulfide-isomerase TRG1 YCL313 PDI1 MFP1 Thioredoxin-related glycoprotein 1 YCL43C YCL043C protein protein yeast Saccharomyces cerevisiae Baker's yeast MKFSAGAVLSWSSLLLASSVFAQQEAVAPEDSAVVKLATDSFNEYIQSHDLVLAEFFAPWCGHCKNMAPEYVKAAETLVEKNITLAQIDCTENQDLCMEHNIPGFPSLKIFKNSDVNNSIDYEGPRTAEAIVQFMIKQSQPAVAVVADLPAYLANETFVTPVIVQSGKIDADFNATFYSMANKHFNDYDFVSAENADDDFKLSIYLPSAMDEPVVYNGKKADIADADVFEKWLQVEALPYFGEIDGSVFAQYVESGLPLGYLFYNDEEELEEYKPLFTELAKKNRGLMNFVSIDARKFGRHAGNLNMKEQFPLFAIHDMTEDLKYGLPQLSEEAFDELSDKIVLESKAIESLVKDFLKGDASPIVKSQEIFENQDSSVFQLVGKNHDEIVNDPKKDVLVLYYAPWCGHCKRLAPTYQELADTYANATSDVLIAKLDHTENDVRGVVIEGYPTIVLYPGGKKSESVVYQGSRSLDSLFDFIKENGHFDVDGKALYEEAQEKAAEEADADAELADEEDAIHDEL 69CF3E05D7F74C94 ero1_yeast Endoplasmic oxidoreductin-1 ERO1 Endoplasmic reticulum oxidoreductase protein 1 YM4987.05C YML130C protein protein yeast Saccharomyces cerevisiae Baker's yeast MRLRTAIATLCLTAFTSATSNNSYIATDQTQNAFNDTHFCKVDRNDHVSPSCNVTFNELNAINENIRDDLSALLKSDFFKYFRLDLYKQCSFWDANDGLCLNRACSVDVVEDWDTLPEYWQPEILGSFNNDTMKEADDSDDECKFLDQLCQTSKKPVDIEDTINYCDVNDFNGKNAVLIDLTANPERFTGYGGKQAGQIWSTIYQDNCFTIGETGESLAKDAFYRLVSGFHASIGTHLSKEYLNTKTGKWEPNLDLFMARIGNFPDRVTNMYFNYAVVAKALWKIQPYLPEFSFCDLVNKEIKNKMDNVISQLDTKIFNEDLVFANDLSLTLKDEFRSRFKNVTKIMDCVQCDRCRLWGKIQTTGYATALKILFEINDADEFTKQHIVGKLTKYELIALLQTFGRLSESIESVNMFEKMYGKRLNGSENRLSSFFQNNFFNILKEAGKSIRYTIENINSTKEGKKKTNNSQSHVFDDLKMPKAEIVPRPSNGTVNKWKKAWNTEVNNVLEAFRFIYRSYLDLPRNIWELSLMKVYKFWNKFIGVADYVSEETREPISYKLDIQ pdia3_human Protein disulfide-isomerase A3 Disulfide isomerase ER-60 Endoplasmic reticulum resident protein 60 58 kDa microsomal protein Endoplasmic reticulum resident protein 57 58 kDa glucose-regulated protein PDIA3 ERP60 GRP58 ERP57 protein protein human Homo sapiens Human MRLRRLALFPGVALLLAAARLAAASDVLELTDDNFESRISDTGSAGLMLVEFFAPWCGHCKRLAPEYEAAATRLKGIVPLAKVDCTANTNTCNKYGVSGYPTLKIFRDGEEAGAYDGPRTADGIVSHLKKQAGPASVPLRTEEEFKKFISDKDASIVGFFDDSFSEAHSEFLKAASNLRDNYRFAHTNVESLVNEYDDNGEGIILFRPSHLTNKFEDKTVAYTEQKMTSGKIKKFIQENIFGICPHMTEDNKDLIQGKDLLIAYYDVDYEKNAKGSNYWRNRVMMVAKKFLDAGHKLNFAVASRKTFSHELSDFGLESTAGEIPVVAIRTAKGEKFVMQEEFSRDGKALERFLQDYFDGNLKRYLKSEPIPESNDGPVKVVVAENFDEIVNNENKDVLIEFYAPWCGHCKNLEPKYKELGEKLSKDPNIVIAKMDATANDVPSPYEVRGFPTIYFSPANKKLNPKKYEGGRELSDFISYLQREATNPPVIQEEKPKKKKKAQEDL pdi1-rnase1-1 501936 N/A 501938 electron donor electron donor unspecified role unspecified role Stoichiometry: 1.0 N/A 501939 electron acceptor electron acceptor unspecified role unspecified role c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false 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 P17967:p.[Cys61Ala;Cys64Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P17967:p.[Cys406Ala;Cys409Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing 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) Stoichiometry: 1.0 enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false 3 A They found that at 3 uM, the N-terminal active site has over a 4-fold defect in the initial rate of oxidation when compared with wild-type PDI (Fig. 3A). In contrast, as seen previously the Cys61Ala, Cys64Ala mutant has a rate of oxidation that is close to that of wild-type PDI, and when the Ero1p concentration is increased modestly (1.3-fold), the initial rates of oxidation can be matched (Fig. 3A). The presence of Ero1p and FAD was required. pdi1-ero1-2 501936 N/A 501939 electron donor electron donor unspecified role unspecified role 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 P17967:p.[Cys61Ala;Cys64Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P17967:p.[Cys406Ala;Cys409Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c-terminal A' sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region A 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 501940 electron acceptor electron acceptor unspecified role unspecified role c-term polyoma Double polyoma epitope MEYMPMEMEYMPME tag tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false 4 A and C, 5 A the rate of oxidation is linearly dependent on PDI concentration. The rate of oxidation of PDI at the indicated concentrations was monitored in the presence of Ero1p (0.3uM) and/or FAD (20uM) the cys406ala, cys409ala mutant appeared to be oxidized more slowly than the cys61ala, cys64ala mutant and they compared their initial rates of oxidation and found that there is a 2-fold disparity in the ability of Ero1p to oxidize the two active sites (Fig. 4C). Additionally, we found that the sum of the initial rates of oxidation of the 2 mutants is equal to the rate of oxidation of PDI strongly suggesting that the two domains are oxidized independently (Fig. 4C). The presence of FAD is required. the rate of oxidation of the A prime domain is half that of the cys61ala, cys64ala mutant indicating that the enhanced rate of oxidation of the A prime domain in the full-length protein is contingent on the presence of the A domain (Fig. 4C). RNase A protects the N-terminal active site of PDI from oxidation by Ero1p and this protection depends on the presence of the substrate binding domains (B prime A prime)(Fig. 5 A). ero1-pdia3 501936 N/A 501941 electron donor electron donor unspecified role unspecified role 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 n-terminal A sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region A' sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecobb Escherichia coli (strain B / BL21) N/A 501940 electron acceptor electron acceptor unspecified role unspecified role c-term polyoma Double polyoma epitope MEYMPMEMEYMPME tag tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false 6 A, B, C the ERp57 active sites lack intrinsic asymmetry, since the rates of oxidation of the individual thioredoxin domains together add up to the rate of oxidation of ERp57. The presence of alkylated RNAse A does not affect the rate of oxidation of ERp57 by Ero1p. The presence of FAD is required. pdi1-rnase1 501937 N/A 501938 electron donor electron donor unspecified role unspecified role N/A 501939 electron acceptor electron acceptor unspecified role unspecified role c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false 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 P17967:p.[Cys61Ala;Cys64Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P17967:p.[Cys406Ala;Cys409Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing 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) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false 2 A and B They followed native disulfide bond formation in refolding RNase A by monitoring its activity using the artificial substrate cCMP. PDI mutant cys61ala-cys64ala and wild-type PDI have a similar ability to catalyze RNase A oxidation (Fig. 2C). Therefore, in comparing cys61ala-cys64ala and wild-type PDI, there is discordance between their rate. of disulfide formation and the yield of native RNase A (Fig. 2, B and C). The re-oxidation of reduced RNaseA(15uM) was assayed by hydrolysis of cCMP in the presence of Ero1p (0.05uM), FAD (100uM). even under conditions in which the rate of disulfide formation is matched, they found that the Cys61-Al, Cys64Ala PDI mutant has a much lower yield of functional RNase A than wild-type PDI (Fig. 3B). Together, the 2 PDI mutants can match the yield of refolded RNase A by wild-type PDI (Fig. 3C). The presence of Erop1 and FAD was required. rnase1-pdia3-1 501937 N/A 501938 electron donor electron donor unspecified role unspecified role N/A 501941 electron acceptor electron acceptor unspecified role unspecified role 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 ecobb Escherichia coli (strain B / BL21) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false 6 E The re-oxidation of reduced RNase A (15 M) was assayed by hydrolysis of cCMP in the presence of Ero1p at the indicated concentrations, FAD. ERp57 is less effective than PDI at isomerization of incorrect disulfides. The presence of Ero1p and FAD was required.