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/ NMR solution structure of the tandem Src homology 3 domains of p47phox complexed with a p22phox-derived proline-rich peptide. NMR solution structure of the tandem Src homology 3 domains of p47phox complexed with a p22phox-derived proline-rich peptide. J. Biol. Chem. (0021-9258) 2006 imex curation Ogura K., Nobuhisa I., Yuzawa S., Takeya R., Torikai S., Saikawa K., Sumimoto H., Inagaki F. BioCreative - Critical Assessment of Information Extraction systems in Biology finagaki@pharm.hokudai.ac.jp 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro biophysical biophysical predetermined predetermined participant predetermined Ogura K., Nobuhisa I., Yuzawa S., Takeya R., Torikai S., Saikawa K., Sumimoto H., Inagaki F. BioCreative - Critical Assessment of Information Extraction systems in Biology In small Angle X-ray Scattering (SAXS) experiments, the sample is irradiated by a well-defined, monochromatic X-ray beam. Structural information of the scattering particles can be derived from the intensity distribution of the scattered beam at very low scattering angles. SAXS data were collected for p47phox-(151-286) in the presence and absence of p22phox-(149-168). The concentration of the proteins was varied from 2 to 16 mg/ml to assess the effects of the protein concentration on the structural parameters. All measurements were made using a SAXS diffractometer in the BL-10C Beamline at the Photon Factory in Tukuba, Japan (25, 26). The wavelength of the x-ray was 1.488 A. We used a 50-l sample cell with quartz windows and a 1-mm path length. The data acquisition time was 300 s for each measurement. Scattering by the protein was determined by subtracting scattering for the solvent (buffer alone) from the scattering for the protein solution. finagaki@pharm.hokudai.ac.jp Accepted 2006-SEP-13 by ORCHARD. 24-JAN-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation NMR solution structure of the tandem Src homology 3 domains of p47phox complexed with a p22phox-derived proline-rich peptide. NMR solution structure of the tandem Src homology 3 domains of p47phox complexed with a p22phox-derived proline-rich peptide. J. Biol. Chem. (0021-9258) 2006 imex curation Ogura K., Nobuhisa I., Yuzawa S., Takeya R., Torikai S., Saikawa K., Sumimoto H., Inagaki F. BioCreative - Critical Assessment of Information Extraction systems in Biology finagaki@pharm.hokudai.ac.jp 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro nmr nuclear magnetic resonance NMR NMR nmr predetermined predetermined participant predetermined Ogura K., Nobuhisa I., Yuzawa S., Takeya R., Torikai S., Saikawa K., Sumimoto H., Inagaki F. BioCreative - Critical Assessment of Information Extraction systems in Biology finagaki@pharm.hokudai.ac.jp Accepted 2006-SEP-13 by ORCHARD. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation NMR solution structure of the tandem Src homology 3 domains of p47phox complexed with a p22phox-derived proline-rich peptide. NMR solution structure of the tandem Src homology 3 domains of p47phox complexed with a p22phox-derived proline-rich peptide. J. Biol. Chem. (0021-9258) 2006 imex curation Ogura K., Nobuhisa I., Yuzawa S., Takeya R., Torikai S., Saikawa K., Sumimoto H., Inagaki F. BioCreative - Critical Assessment of Information Extraction systems in Biology finagaki@pharm.hokudai.ac.jp 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro pull down pull down predetermined predetermined participant predetermined Ogura K., Nobuhisa I., Yuzawa S., Takeya R., Torikai S., Saikawa K., Sumimoto H., Inagaki F. BioCreative - Critical Assessment of Information Extraction systems in Biology finagaki@pharm.hokudai.ac.jp Accepted 2006-SEP-13 by ORCHARD. 24-JAN-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation NMR solution structure of the tandem Src homology 3 domains of p47phox complexed with a p22phox-derived proline-rich peptide. NMR solution structure of the tandem Src homology 3 domains of p47phox complexed with a p22phox-derived proline-rich peptide. J. Biol. Chem. (0021-9258) 2006 imex curation Ogura K., Nobuhisa I., Yuzawa S., Takeya R., Torikai S., Saikawa K., Sumimoto H., Inagaki F. BioCreative - Critical Assessment of Information Extraction systems in Biology finagaki@pharm.hokudai.ac.jp 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro fluorescence spectr classical fluorescence spectroscopy fluorescence spectr predetermined predetermined participant predetermined Ogura K., Nobuhisa I., Yuzawa S., Takeya R., Torikai S., Saikawa K., Sumimoto H., Inagaki F. BioCreative - Critical Assessment of Information Extraction systems in Biology The binding affinity of p22phox peptides for p47phox-(151-286) was monitored by fluorescence spectroscopy using a Shimadzu RF-5300PC fluorometer (excitation at 295 nm and emission at 330 nm). Aliquots of synthetic p22phox peptides were incubated for 2 h with 3.0M p47phox-(151-286) in 20 mM sodium phosphate buffer, pH 7.0. All measurements were performed at 25C. Dissociation constants (Kd) were determined by nonlinear regression analysis using Kaleidagraph (Synergy Software) assuming a simple bimolecular association. finagaki@pharm.hokudai.ac.jp Accepted 2006-SEP-13 by ORCHARD. 24-JAN-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation NMR solution structure of the tandem Src homology 3 domains of p47phox complexed with a p22phox-derived proline-rich peptide. NMR solution structure of the tandem Src homology 3 domains of p47phox complexed with a p22phox-derived proline-rich peptide. J. Biol. Chem. (0021-9258) 2006 imex curation Ogura K., Nobuhisa I., Yuzawa S., Takeya R., Torikai S., Saikawa K., Sumimoto H., Inagaki F. BioCreative - Critical Assessment of Information Extraction systems in Biology finagaki@pharm.hokudai.ac.jp 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro nmr nuclear magnetic resonance NMR NMR nmr predetermined predetermined participant predetermined Ogura K., Nobuhisa I., Yuzawa S., Takeya R., Torikai S., Saikawa K., Sumimoto H., Inagaki F. BioCreative - Critical Assessment of Information Extraction systems in Biology To identify the region of p22phox essential for p47phox binding in solution, they performed NMR titration experiments using two synthetic peptides, p22phox-(149�162) and p22phox-(149�168). Aliquots of each peptide solution were added to the sample solution of the 15N-labeled tandem SH3 domains of p47phox, and the two-dimensional 1H-15N HSQC spectra were analyzed. finagaki@pharm.hokudai.ac.jp Accepted 2006-SEP-13 by ORCHARD. 24-JAN-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation ncf1_human Neutrophil cytosol factor 1 NCF1 Neutrophil NADPH oxidase factor 1 47 kDa neutrophil oxidase factor p47-phox NCF-47K 47 kDa autosomal chronic granulomatous disease protein Nox-organizing protein 2 Nox organizer 2 SH3 and PX domain-containing protein 1A NOXO2 SH3PXD1A protein protein human Homo sapiens Human MGDTFIRHIALLGFEKRFVPSQHYVYMFLVKWQDLSEKVVYRRFTEIYEFHKTLKEMFPIEAGAINPENRIIPHLPAPKWFDGQRAAENRQGTLTEYCSTLMSLPTKISRCPHLLDFFKVRPDDLKLPTDNQTKKPETYLMPKDGKSTATDITGPIILQTYRAIANYEKTSGSEMALSTGDVVEVVEKSESGWWFCQMKAKRGWIPASFLEPLDSPDETEDPEPNYAGEPYVAIKAYTAVEGDEVSLLEGEAVEVIHKLLDGWWVIRKDDVTGYFPSMYLQKSGQDVSQAQRQIKRGAPPRRSSIRNAHSIHQRSRKRLSQDAYRRNSVRFLQQRRRQARPGPQSPGSPLEEERQTQRSKPQPAVPPRPSADLILNRCSESTKRKLASAV 3D91EDC99A1B6417 cy24a_human Cytochrome b-245 light chain p22 phagocyte B-cytochrome Neutrophil cytochrome b 22 kDa polypeptide p22-phox Cytochrome b(558) alpha chain Cytochrome b558 subunit alpha Superoxide-generating NADPH oxidase light chain subunit CYBA protein protein human Homo sapiens Human MGQIEWAMWANEQALASGLILITGGIVATAGRFTQWYFGAYSIVAGVFVCLLEYPRGKRKKGSTMERWGQKYMTAVVKLFGPFTRNYYVRAVLHLLLSVPAGFLLATILGTACLAIASGIYLLAAVRGEQWTPIEPKPRERPQIGGTIKQPPSNPPPRPPAEARKKPSEEEAAVAAGGPPGGPQVNPIPVTDEVV DIP protein P13498 original sequence version: 99 428427AD19398240 ncf1-cyba 2 7 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobb Escherichia coli (strain B / BL21) 6 unspecified role unspecified role neutral component neutral component region tandem SH3 domains without PBR/AIR sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region 15n label 15n label 15N N15 15N N15 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region 13c label 13c label C13 13C C13 13C undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction There are two extra residues at the N-terminus of the p47phox151-286 (NCF1) construct, that is GS, as compared with the Uniprot entry. These are detailed in the pdb entry 1WLP. These are presumably due to removal of the N-terminal glutathione S-transferase tag by thrombin protease. The recombinant p22phox149-168 contains 5 additional residues derived from the protease cleavage site to remove the GST tag (GPLGS). This is detailed in the pdb entry 1WLP. The solution structure of p47phox-(151�286) complexed with p22phox-(149�168) was determined using an ARIA/CNS protocol. 4 and Table 1, Fig. 3 A and B They measured the hetNOE spectra of the free and complexed forms of p47phox151-286 by performing a series of NMR experiments. These findings indicate that the 2 SH3 domains can move freeely in the free state, but that on binding to the peptide, both undergo structural rearrangements causing the complex to become rigid. This is consistent with the SAXS data which show that the tandem SH3 domains have an extended structure in the free state but a compact structure in the complex state. ncf1-cyba-2 5 6 unspecified role unspecified role neutral component neutral component region 15n label 15n label 15N N15 15N N15 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region tandem SH3 domains without PBR/AIR sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobb Escherichia coli (strain B / BL21) 7 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) direct interaction direct interaction There are two extra residues at the N-terminus of the p47phox151-286 (NCF1) construct, that is GS, as compared with the Uniprot entry. These are detailed in the pdb entry 1WLP. These are presumably due to removal of the N-terminal glutathione S-transferase tag by thrombin protease. 1 A, B Aliquots of each peptide solution were added to the sample solution of the 15N-labeled tandem SH3 domains of p47phox, and the two-dimensional 1H-15N HSQC spectra were analyzed (Fig. 1). We found that many of the signals decreased and finally disappeared as the concentration of p22phox-(149�162) increased (Fig. 1a). Notably, three of five tryptophan signals disappeared, leaving only two observed signals (indicated by asterisks in Fig. 1a). This shows that a structural change occurs in the tandem SH3 domains upon complex formation. This produces an intermediate slow-exchange limit in the NMR spectra so that some signals cannot be observed because of exchange broadening. In contrast, an increase in concentration of p22phox-(149 �168) caused changes in the chemical shifts for a number of resonance As the concentration of p22phox-(149�162) increased, three of five tryptophan signals disappeared, leaving only two observed signals. This shows that a structural change occurs in the tandem SH3 domains upon complex formation. During titration with p22phox-(149-168),all five tryptophan signals, which consist of two Trp residues in the Nterminal SH3 domain and three Trp residues in the C-terminal SH3 domain, showed significant changes in their chemical, indicating that p22phox-(149 �168) stably interacts with both SH3 domains. ncf1-cyba-3 4 7 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) 6 unspecified role unspecified role neutral component neutral component region tandem SH3 domains without PBR/AIR sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction There are two extra residues at the N-terminus of the p47phox151-286 (NCF1) construct, that is GS, as compared with the Uniprot entry. These are detailed in the pdb entry 1WLP. These are presumably due to removal of the N-terminal glutathione S-transferase tag by thrombin protease. 1 D The Kd values for complex formation between p47phox-(151�286) and the two p22phox peptides were subsequently determined by fluorescence titration method. The fluorescence data gave Kd values of 8.67 M for p22phox-(149�162) and 0.64 M for p22phox-(149�168), respectively (Fig. 1, c and d), indicating that residues 163�168 of p22phox enhance the binding affinity of p22phox for p47phox by more than 10-fold. The fluorescence data gave Kd values of 8.67 E-6 M for p22phox-(149-162) and 6.4 E-5M for p22phox-(149-168), respectively (Fig. 1, c and d), indicating that residues 163-168 of p22phox enhance the binding affinity of p22phox for p47phox by more than 10-fold. ncf1-cyba-4 4 7 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) 6 unspecified role unspecified role neutral component neutral component region tandem SH3 domians without PBR/AIR sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction There are two extra residues at the N-terminus of the p47phox151-286 (NCF1) construct, that is GS, as compared with the Uniprot entry. These are detailed in the pdb entry 1WLP. These are presumably due to removal of the N-terminal glutathione S-transferase tag by thrombin protease. 1 C The Kd values for complex formation between p47phox-(151�286) and the two p22phox peptides were subsequently determined by fluorescence titration method. The fluorescence data gave Kd values of 8.67 M for p22phox-(149�162) and 0.64 M for p22phox-(149�168), respectively (Fig. 1, c and d), indicating that residues 163�168 of p22phox enhance the binding affinity of p22phox for p47phox by more than 10-fold. The fluorescence data gave Kd values of 8.67 E-6 M for p22phox-(149-162) and 6.4 E-5 M for p22phox-(149-168), respectively (Fig. 1, c and d), indicating that residues 163-168 of p22phox enhance the binding affinity of p22phox for p47phox by more than 10-fold. ncf1-cyba-5 1 7 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) 6 unspecified role unspecified role neutral component neutral component region tandem SH3 domains without PBR/AIR sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction There are two extra residues at the N-terminus of the p47phox151-286 (NCF1) construct, that is GS, as compared with the Uniprot entry. These are detailed in the pdb entry 1WLP. These are presumably due to removal of the N-terminal glutathione S-transferase tag by thrombin protease. 2 A and B In contrast, the P(r) for the complex showed a peak at 20� and a skirt extending up to 60� (Fig. 2b). From these findings, we concluded that p47phox-(151�286) has a compact globular structure when complexed with p22phox-(149�168), whereas the peptide-free form has an extended structure. From these findings, they concluded that p47phox-(151�286) has a compact globular structure when complexed with p22phox-(149�168), whereas the peptide-free form has an extended structure. Consistent with the P(r) analysis, Guinier analysis determined that the radius of gyration is 25.3 � for the free form and 19.5 � for the complex form. ncf1-cyba-6 3 6 unspecified role unspecified role prey prey region tandem SH3 domains without PBR/AIR sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined P14598:p.Phe209Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain F A Wed May 02 16:13:27 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe209ala' Tue Jun 30 19:20:20 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe209Ala' P14598:p.Ile164Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain I A Wed May 02 17:05:06 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ile164ala' Tue Jun 30 19:37:43 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ile164Ala' ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role bait bait region alpha-helical region C-terminal to PRR sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined [EBI-986424]Certain and ragged-n-terminus positions must always be strictly superior to 0 and inferior or equal to the protein sequence length. Actual positions : 196-196, sequence length 195 [EBI-986424]133-196 P13498:p.Ala163_Arg164delinsGlyGly mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain AR GG Fri Jul 22 11:02:50 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'ala163gly,arg164gly' Wed May 02 14:34:27 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ala_arg163-164gly_gly' Tue Jun 30 15:48:07 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ala163_Arg164delinsGlyGly' region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction 8 The authors state that the I164A substitution in the p47 phox construct causes a more impaired interaction than the F209A substitution. However, in Fig. 8 no band can be seen for the I164A mutation for the interaction with the wild-type. Also the authors state the mutant p47phox proteins interact more weakly with the mutant p22phox protein, but again no bands can be seen. As shown in Fig. 8, the A163G/R164G substitution led to decreased binding to p47phox-(151�286), implying a significant role for the p22phox -helical region. Because the -helix appears to interact directly with Ile164 and Phe209 in the p47phox N- SH3 (Fig. 7a), we next investigated the roles of these residues. For this purpose, we prepared mutants of p47phox-(151�286) carrying substitutions of alanine for Ile164 or Phe209 and tested their ability to bind to p22phox. Compared with the wild-type p47phox-(151�286), the mutant protein with the F209A substitution weakly bound to p22phox (Fig. 8). The I164A substitution resulted in a more impaired interaction with p22phox (Fig. 8), which is consistent with the present finding that Ile164 makes stronger contacts with the p22phox -helix than does Phe209 (Fig. 7a). These mutant p47phox proteins also interacted more weakly with the -helix-defective p22phox (A163G/R164G) than with the wild-type p22phox These results indicate that the alpha-helical conformation of the region C-terminal to the p22phox PRR (amino acids 161�164) participates in the full interaction with p47phox-(151�286).