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/ ahuja-2009-1 Analyzing the interaction of RseA and RseB, the two negative regulators of the sigmaE envelope stress response, using a combined bioinformatic and experimental strategy. in vitro In vitro pull down pull down predetermined predetermined participant predetermined Ahuja N., Korkin D., Chaba R., Cezairliyan B.O., Sauer R.T., Kim K.K., Gross C.A. They were unable to perform similar experiments with the N-terminal domain of RseB because that domain alone forms high molecular weight (MW) aggregates. The Rse-A and the RseB constructs that were used for the pull-down were previously purified cgross@cgl.ucsf.edu J. Biol. Chem. (0021-9258) 2009 Only protein-protein interactions imex curation ahuja-2009-2 Analyzing the interaction of RseA and RseB, the two negative regulators of the sigmaE envelope stress response, using a combined bioinformatic and experimental strategy. ecoli Escherichia coli (strain K12) crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Ahuja N., Korkin D., Chaba R., Cezairliyan B.O., Sauer R.T., Kim K.K., Gross C.A. They were unable to perform similar experiments with the N-terminal domain of RseB because that domain alone forms high molecular weight (MW) aggregates. The Rse-A and the RseB constructs that were used for the pull-down were previously purified cgross@cgl.ucsf.edu J. Biol. Chem. (0021-9258) 2009 Only protein-protein interactions imex curation rsea_ecoli Anti-sigma-E factor RseA yfiJ rseA mclA b2572 JW2556 Regulator of SigE Sigma-E anti-sigma factor RseA Sigma-E factor negative regulatory protein protein protein ecoli Escherichia coli (strain K12) MQKEQLSALMDGETLDSELLNELAHNPEMQKTWESYHLIRDSMRGDTPEVLHFDISSRVMAAIEEEPVRQPATLIPEAQPAPHQWQKMPFWQKVRPWAAQLTQMGVAACVSLAVIVGVQHYNGQSETSQQPETPVFNTLPMMGKASPVSLGVPSEATANNGQQQQVQEQRRRINAMLQDYELQRRLHSEQLQFEQAQTQQAAVQVPGIQTLGTQSQ rseb_ecoli Sigma-E factor regulatory protein RseB rseB b2571 JW2555 protein protein ecoli Escherichia coli (strain K12) MKQLWFAMSLVTGSLLFSANASATPASGALLQQMNLASQSLNYELSFISINKQGVESLRYRHARLDNRPLAQLLQMDGPRREVVQRGNEISYFEPGLEPFTLNGDYIVDSLPSLIYTDFKRLSPYYDFISVGRTRIADRLCEVIRVVARDGTRYSYIVWMDTESKLPMRVDLLDRDGETLEQFRVIAFNVNQDISSSMQTLAKANLPPLLSVPVGEKAKFSWTPTWLPQGFSEVSSSRRPLPTMDNMPIESRLYSDGLFSFSVNVNRATPSSTDQMLRTGRRTVSTSVRDNAEITIVGELPPQTAKRIAENIKFGAAQ rsea-rseb 1320000 n/a 1320002 unspecified role unspecified role bait bait nucl transformation nucleic acid transformation nucl transformation histag 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 region 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) n/a 1320003 unspecified role unspecified role prey prey nucl transformation nucleic acid transformation nucl transformation region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.Gln182Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.[Asp245_Asn246delinsAlaAla;Ile249_Glu250delinsAlaAla] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.Ile249_Glu250delinsAlaAla mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.Asp176Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.Ile249Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.Asp171Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.Arg238_Arg239delinsAlaAla mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false flag-tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range false P0AFX9:p.Glu250Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.Glu178Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) 1320009 1320007 direct interaction direct interaction false false false Figure 2 A ,C, 5A, 6A When N-terminally Flag-tagged C-terminal domain of RseB (residues 218 –318) was allowed to interact with N-terminally His tagged RseA-peri (121-216), only about 15% of the C-terminal domain of RseB (Fig.2B, C) was bound to RseA-peri. rsea-rseb-1 1320000 n/a 1320003 unspecified role unspecified role prey prey nucl transformation nucleic acid transformation nucl transformation flag-tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region 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) n/a 1320002 unspecified role unspecified role bait bait nucl transformation nucleic acid transformation nucl transformation P0AFX7:p.Leu177Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Asp179Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Gln183Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Arg185Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Asn174Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Glu181Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Leu182Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false trx tag fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false P0AFX7:p.Met176Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Tyr180Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Leu186Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Ser188Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false histag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false P0AFX7:p.Gln178Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.His187Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) 1320029 1320023 direct interaction direct interaction false false false Figure 4, 7 rsea-rseb-3 1320000 n/a 1320003 unspecified role unspecified role prey prey P0AFX9:p.Arg238_Arg239delinsAlaAla mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.[Asp245_Asn246delinsAlaAla;Ile249_Glu250delinsAlaAla] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.Ile249_Glu250delinsAlaAla mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.Glu181Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.Ile249Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.Glu250Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.Asp174Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.Asp176Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.Glu178Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.Gln182Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false flag-tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P0AFX9:p.Asp171Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1320002 unspecified role unspecified role bait bait histag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region 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) 1320058 1320054 direct interaction direct interaction false false false Figures 5B, 6B rsea-rseb-2 1320000 n/a 1320002 unspecified role unspecified role bait bait nucl transformation nucleic acid transformation nucl transformation P0AFX7:p.Leu186Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false histag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false trx tag fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false P0AFX7:p.Tyr180Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Leu182Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Ser188Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Asn174Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Met176Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.His187Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Arg185Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Gln178Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Glu189Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Glu181Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Gln183Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Ile173Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Leu177Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Asp179Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1320003 unspecified role unspecified role prey prey nucl transformation nucleic acid transformation nucl transformation P0AFX9:p.Ile249_Glu250delinsAlaAla mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false flag-tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) 1320081 1320065 direct interaction direct interaction false false false Figure 7 rsea-rseb-4 1320001 n/a 1320003 unspecified role unspecified role prey prey nucl transformation nucleic acid transformation nucl transformation region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false flag-tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range false P0AFX9:p.Pro248Cys mutation mutation certain certain sequence position certain certain certain sequence position certain false n/a 1320002 unspecified role unspecified role bait bait nucl transformation nucleic acid transformation nucl transformation histag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false P0AFX7:p.Leu186Cys mutation increasing strength mutation increasing interaction strength mutation increasing strength certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Asp179Cys mutation increasing strength mutation increasing interaction strength mutation increasing strength certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Ile173Cys mutation increasing strength mutation increasing interaction strength mutation increasing strength certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false 1320093 1320085 physical association physical association false false false Figure 8 rsea-rseb-5 1320001 n/a 1320002 unspecified role unspecified role bait bait nucl transformation nucleic acid transformation nucl transformation P0AFX7:p.His187Cys mutation increasing strength mutation increasing interaction strength mutation increasing strength certain certain sequence position certain certain certain sequence position certain false P0AFX7:p.Asp179Cys mutation increasing strength mutation increasing interaction strength mutation increasing strength certain certain sequence position certain certain certain sequence position certain false histag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n/a 1320003 unspecified role unspecified role prey prey nucl transformation nucleic acid transformation nucl transformation flag-tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range false P0AFX9:p.Ser251Cys mutation mutation certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false 1320103 1320099 physical association physical association false false false Figure 8