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/ sohn-2007-1 Allosteric Activation of DegS, a Stress Sensor PDZ Protease. in vitro In vitro protease assay protease assay predetermined predetermined participant predetermined Sohn J., Grant RA., Sauer RT. Cleavage reactions were initiated by addition of enzyme or substrate to reaction buffer with or without OMP peptide. At each time pointan aliquot of the cleavage reaction was mixed with 10% TCA. Insoluble full-length substrate and the C-terminal cleavage product were separated from the soluble N-terminal product by centrifugation and extent of cleavage determined by scintiallation counting. bobsauer@mit.edu Cell (0092-8674) 2007 Only protein-protein interactions 6 imex curation sohn-2007-2 Allosteric Activation of DegS, a Stress Sensor PDZ Protease. in vitro In vitro protease assay protease assay predetermined predetermined participant predetermined Sohn J., Grant RA., Sauer RT. Cleavage performed and products then separated by SDS-PAGE bobsauer@mit.edu Cell (0092-8674) 2007 Only protein-protein interactions 6 imex curation Cleavage reactions were initiated by addition of enzyme or substrate to reaction buffer with or without OMP peptide. At each time pointan aliquot of the cleavage reaction was mixed with 10% TCA. Insoluble full-length substrate and the C-terminal cleavage product were separated from the soluble N-terminal product by centrifugation and extent of cleavage determined by scintiallation counting. sohn-2007-3 Allosteric Activation of DegS, a Stress Sensor PDZ Protease. in vitro In vitro fps fluorescence polarization spectroscopy Fluorescence anisotropy FPS FPS Fluorescence anisotropy fps predetermined predetermined participant predetermined Sohn J., Grant RA., Sauer RT. Binding experiments monitored by fluorescence anisotropy (excitation 480 nm; emission 520 nm) were performed using a PTI QM-2000-4SE spectrofluorimeter bobsauer@mit.edu Cell (0092-8674) 2007 Only protein-protein interactions 6 imex curation Cleavage reactions were initiated by addition of enzyme or substrate to reaction buffer with or without OMP peptide. At each time pointan aliquot of the cleavage reaction was mixed with 10% TCA. Insoluble full-length substrate and the C-terminal cleavage product were separated from the soluble N-terminal product by centrifugation and extent of cleavage determined by scintiallation counting. sohn-2007-4 Allosteric Activation of DegS, a Stress Sensor PDZ Protease. in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Sohn J., Grant RA., Sauer RT. bobsauer@mit.edu Cell (0092-8674) 2007 Only protein-protein interactions 6 imex curation Cleavage reactions were initiated by addition of enzyme or substrate to reaction buffer with or without OMP peptide. At each time pointan aliquot of the cleavage reaction was mixed with 10% TCA. Insoluble full-length substrate and the C-terminal cleavage product were separated from the soluble N-terminal product by centrifugation and extent of cleavage determined by scintiallation counting. degs_ecoli Serine endoprotease DegS degS hhoB htrH b3235 JW3204 Site-1 protease DegS Site-1-type intramembrane protease protein protein ecoli Escherichia coli (strain K12) MFVKLLRSVAIGLIVGAILLVAMPSLRSLNPLSTPQFDSTDETPASYNLAVRRAAPAVVNVYNRGLNTNSHNQLEIRTLGSGVIMDQRGYIITNKHVINDADQIIVALQDGRVFEALLVGSDSLTDLAVLKINATGGLPTIPINARRVPHIGDVVLAIGNPYNLGQTITQGIISATGRIGLNPTGRQNFLQTDASINHGNSGGALVNSLGELMGINTLSFDKSNDGETPEGIGFAIPFQLATKIMDKLIRDGRVIRGYIGIGGREIAPLHAQGGGIDQLQGIVVNEVSPDGPAANAGIQVNDLIISVDNKPAISALETMDQVAEIRPGSVIPVVVMRDDKQLTLQVTIQEYPATN 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 yyf peptide DNRDGNVYYF peptide peptide polypeptide oligopeptide oligopeptide polypeptide in vitro In vitro DNRDGNVYYF An amino acid sequence which is not represented in UniProt degs-rsea-1 Cleavage of RseA by DegS 680201 N/A 680205 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain range range false range range certain certain sequence position certain false ecobb Escherichia coli (strain B / BL21) N/A 680206 enzyme target enzyme target substrate substrate neutral component neutral component c-terminal 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 periplasmic region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecobb Escherichia coli (strain B / BL21) 680214 680211 protein cleavage protein cleavage false false false Km = 570 +/- 50 uM Vmax = 0.8 +/- 0.1 s-1 Trimeric delta-DegS-1 Hill constant = 1.8 +/- 0.2 Fig. 1D This construct (delta-DegS) is lacking the PDZ domain which appears to cause in inhibition of the reaction which is relieved by the addition of peptide. degs-rsea-3 Cleavage of RseA by DegS, YYF peptide as stimulator 680201 N/A 680205 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false undefined mutation mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false ecobb Escherichia coli (strain B / BL21) N/A 680206 enzyme target enzyme target substrate substrate neutral component neutral component c-terminal 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 periplasmic region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecobb Escherichia coli (strain B / BL21) N/A 680207 stimulator stimulator neutral component neutral component protein cleavage protein cleavage false false false Fig. 1B, 1C, 1D, 1E Km = 750 +/- 120 uM Vmax = 1.1 +/- 0.2 s-1, trimeric DegS-1 Hill constant 1.6 +/- 0.2 degs-rsea-4 Cleavage of RseA by DegS 680201 N/A 680206 enzyme target enzyme target substrate substrate neutral component neutral component c-terminal 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 periplasmic region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false asn216asn_tyr_asn_ph fusion protein fusion protein certain certain sequence position certain certain certain sequence position certain false ecobb Escherichia coli (strain B / BL21) N/A 680205 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P0AEE3:p.Tyr162Ala mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false P0AEE3:p.Tyr162Phe mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false P0AEE3:p.Lys243Asp mutation increasing mutation increasing interaction mutation increasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false P0AEE3:p.Arg256Ala mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false P0AEE3:p.Arg256Asp mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false P0AEE3:p.Asp320Ala mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false undefined mutation mutation increasing mutation increasing interaction mutation increasing insertion analysis insertion analysis certain certain sequence position certain certain certain sequence position certain false P0AEE3:p.Asp122Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false P0AEE3:p.Arg178Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false P0AEE3:p.Glu227Ala mutation increasing mutation increasing interaction mutation increasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false ecobb Escherichia coli (strain B / BL21) protein cleavage protein cleavage false false false Fig. 1F, 4A, 4B, Table 3 Km = 33 +/- 5 uM Fusing the C-terminal tripeptide of E. coli OmpC (YQF) to the C terminus of RseA may mean the fusion substrate would bind more tightly by tethering itself to the PDZ domain of DegS and also be self activating. In fact, the fusion substrate interacts with DegS in a manner that precludes favorable substrate-substrate interactions. degs-rsea-2 Cleavage of RseA by DegS 680202 N/A 680206 enzyme target enzyme target substrate substrate neutral component neutral component c-terminal 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 periplasmic region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecobb Escherichia coli (strain B / BL21) N/A 680205 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 region 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 680207 stimulator stimulator neutral component neutral component protein cleavage protein cleavage false false false Fig. 1A degs-yyf_peptide-1 Peptide binding of DegS 680203 N/A 680205 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 pdz region sufficient to bind sufficient binding region sufficient to bind range range range range false ecobb Escherichia coli (strain B / BL21) N/A 680207 stimulator stimulator neutral component neutral component protein cleavage protein cleavage false false false Fig. 2B Kd full-length DegS = 4.6E-6M dgs-1 Trimerization of DegS demonstrated by X-ray crystallogrphy 680204 N/A 680205 unspecified role unspecified role neutral component neutral component domain 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: 3.0 direct interaction direct interaction false false false Protein is selenomethionine-substituted 2.04-2.5A