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/ fox-2016-3 in vitro In vitro fps fluorescence polarization spectroscopy Fluorescence anisotropy FPS FPS Fluorescence anisotropy fps tag fluorescence tag visualisation by fluorescence tag fluorescence Fox JC., Tyler RC., Peterson FC., Dyer DP., Zhang F., Linhardt RJ., Handel TM., Volkman BF. bvolkman@mcw.edu Biochemistry 2016 imex curation Only protein-protein interactions imex curation fox-2016-2 in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Fox JC., Tyler RC., Peterson FC., Dyer DP., Zhang F., Linhardt RJ., Handel TM., Volkman BF. The Kd values obtained were calculated through both kinetic and steady state analysis. Values obtained by each method for each mutant vary considerably. Figures 5A/B appear to show increased binding of the R9A and K25A mutants, but Kd values obtained in Table 2 do not unambiguously support this fact. Alanine point mutations of several basic residues were made within the W55D background. All variants except CC0, CC3, and WT were made in the W55D background. Several XCL1 variants were eluted from a heparin Sepharose with increasing NaCl concentrations. To assess the heparin binding affinity for each XCL1 protein, the nonspecific electrostatic attractions measured by cation exchange were subtracted from the values measured by heparin Sepharose chromatography bvolkman@mcw.edu Biochemistry 2016 imex curation Only protein-protein interactions imex curation fox-2016-1 in vitro In vitro affinity chrom affinity chromatography technology Affinity purification Affinity purification affinity chrom predetermined predetermined participant predetermined Fox JC., Tyler RC., Peterson FC., Dyer DP., Zhang F., Linhardt RJ., Handel TM., Volkman BF. Alanine point mutations of several basic residues were made within the W55D background. All variants except CC0, CC3, and WT were made in the W55D background. Several XCL1 variants were eluted from a heparin Sepharose column with increasing NaCl concentrations. To assess the heparin binding affinity for each XCL1 protein, the nonspecific electrostatic attractions measured by cation exchange were subtracted from the values measured by heparin Sepharose chromatography bvolkman@mcw.edu Biochemistry 2016 imex curation Only protein-protein interactions imex curation p47992-pro_0000005248 Lymphotactin XCL1 LTN SCYC1 ATAC C motif chemokine 1 Cytokine SCM-1 Lymphotaxin SCM-1-alpha Small-inducible cytokine C1 XC chemokine ligand 1 protein protein human Homo sapiens Human VGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDVVRSMDRKSNTRNNMIQTKPTGTQQSTNTAVTLTG 22 114 heparin-dp4 small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) dp8 dp8 small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) hs-10 HS-10 small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) hs-14 HS-14 small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) hs-15 HS-15 small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) heparin Heparin small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) heparin-xcl1-3 1936265 N/A 1936268 unspecified role unspecified role neutral component neutral component P47992-PRO_0000005248:p.Trp55Asp mutation mutation certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Arg23Ala;Arg43Ala;Trp55Asp] mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 N/A 1936269 unspecified role unspecified role neutral component neutral component region fluorescein fluorescein label fluorescein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false direct interaction direct interaction false false false 1936265 Figure 6B,7 sodium chloride heparin_dp4-xcl1-1 1936265 N/A 1936269 unspecified role unspecified role neutral component neutral component region fluorescein fluorescein label fluorescein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 1936268 unspecified role unspecified role neutral component neutral component P47992-PRO_0000005248:p.Trp55Asp mutation mutation certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 N/A 1936269 competitor competitor competitor neutral component neutral component direct interaction direct interaction false false false 1936265 1936265 Figure 6C heparin_dp4-heparin_dp4-1 1936265 N/A 1936269 unspecified role unspecified role neutral component neutral component region fluorescein fluorescein label fluorescein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 1936268 unspecified role unspecified role neutral component neutral component P47992-PRO_0000005248:p.Trp55Asp mutation mutation certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 N/A 1936270 competitor competitor competitor neutral component neutral component direct interaction direct interaction false false false 1936265 1936265 Figure 6C-2 heparin_dp4-heparin_dp4-2 1936265 N/A 1936269 unspecified role unspecified role neutral component neutral component region fluorescein fluorescein label fluorescein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 1936268 unspecified role unspecified role neutral component neutral component P47992-PRO_0000005248:p.Trp55Asp mutation mutation certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 N/A 1936271 competitor competitor competitor neutral component neutral component direct interaction direct interaction false false false 1936265 1936265 Figure 7 An unsulfated heparan molecule and a less sulfated molecule were unable to compete for the binding sites occupied by the dp4 probe. heparin_dp4-hs_10-1 1936265 N/A 1936269 unspecified role unspecified role neutral component neutral component region fluorescein fluorescein label fluorescein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 1936268 unspecified role unspecified role neutral component neutral component P47992-PRO_0000005248:p.Trp55Asp mutation mutation certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 N/A 1936272 competitor competitor competitor neutral component neutral component direct interaction direct interaction false false false 1936265 1936265 Figure 7-2 An unsulfated heparan molecule and a less sulfated molecule were unable to compete for the binding sites occupied by the dp4 probe. heparin_dp4-hs_14-1 1936265 N/A 1936269 unspecified role unspecified role neutral component neutral component region fluorescein fluorescein label fluorescein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 1936268 unspecified role unspecified role neutral component neutral component P47992-PRO_0000005248:p.Trp55Asp mutation mutation certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 N/A 1936273 competitor competitor competitor neutral component neutral component direct interaction direct interaction false false false 1936265 1936265 Figure 7-3 An unsulfated heparan molecule and a less sulfated molecule were unable to compete for the binding sites occupied by the dp4 probe. heparin-xcl1-2 1936266 N/A 1936268 unspecified role unspecified role prey prey P47992-PRO_0000005248:p.Trp55Asp mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Arg9Ala;Trp55Asp] mutation mutation certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Lys25Ala;Trp55Asp] mutation mutation certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Val21Cys;Val59Cys] mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Arg23Ala;Arg43Ala;Trp55Asp] mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false 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: 2.0 N/A 1936274 unspecified role unspecified role bait bait pig Sus scrofa (Pig) Pig direct interaction direct interaction false false false Figure 5, Table 2 A monomeric form of XCL1 (CC3) also showed weaker binding ; CC3 is an XCL1 double mutant (V21C, V59C) containing a second disulfide bond which restricts the protein to a monomeric form. The Kd values obtained were calculated through both kinetic and steady state analysis. Values obtained by each method for each mutant vary considerably. heparin-xcl1-4 1936266 N/A 1936268 unspecified role unspecified role prey prey P47992-PRO_0000005248:p.[Val21Cys;Val59Cys] mutation mutation 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 N/A 1936274 unspecified role unspecified role bait bait pig Sus scrofa (Pig) Pig Stoichiometry: 1.0 direct interaction direct interaction false false false Figure 5i, Table 2 A monomeric form of XCL1 (CC3) also showed weaker binding ; CC3 is an XCL1 double mutant (V21C, V59C) containing a second disulfide bond which restricts the protein to a monomeric form. The Kd values obtained were calculated through both kinetic and steady state analysis. Values obtained by each method for each mutant vary considerably. heparin-xcl1-1 1936267 N/A 1936268 unspecified role unspecified role prey prey P47992-PRO_0000005248:p.Trp55Asp 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 P47992-PRO_0000005248:p.[Trp55Asp;Arg70Ala] mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Arg9Ala;Trp55Asp] mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Arg18Ala;Trp55Asp] mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Arg23Ala;Trp55Asp] mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Arg35Ala;Trp55Asp] mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Lys42Ala;Trp55Asp] mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Lys25Ala;Trp55Asp] mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Arg43Ala;Trp55Asp] mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Lys8Ala;Trp55Asp] mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Val21Cys;Val59Cys] mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Arg23Ala;Arg43Ala;Trp55Asp] mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Lys46Ala;Trp55Asp] mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Trp55Asp;Arg61Ala] mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P47992-PRO_0000005248:p.[Cys11Ala;Cys48Ala] mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength 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: 2.0 N/A 1936274 unspecified role unspecified role bait bait pig Sus scrofa (Pig) Pig direct interaction direct interaction false false false 3, 4A, Table 1 An unfolded/unstructured form of XCL1 showed substantially reduced binding; the CCO mutant has a double mutation of C11 and C48 to alanine which removes an intermolecular disulfide bond. A monomeric form of XCL1 also showed little binding;CC3 is an XCL1 double mutant (V21C, V59C) containing a second disulfide bond which restricts the protein to a monomeric form.