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/ rha-2009-1 Multiple binding modes between HNF4alpha and the LXXLL motifs of PGC-1alpha lead to full activation. in vitro In vitro pull down pull down autoradiography autoradiography Rha G.B., Wu G., Shoelson S.E., Chi Y.I. Ulcerative colitis - Interactions of proteins identified as having a link to ulcerative colitis ychi@uky.edu J. Biol. Chem. (0021-9258) 2009 Only protein-protein interactions imex curation IBD - Inflammatory bowel disease rha-2009-2 Multiple binding modes between HNF4alpha and the LXXLL motifs of PGC-1alpha lead to full activation. in vitro In vitro itc isothermal titration calorimetry ITC ITC itc predetermined predetermined participant predetermined Rha G.B., Wu G., Shoelson S.E., Chi Y.I. Ulcerative colitis - Interactions of proteins identified as having a link to ulcerative colitis Affinities calculated from the ITC assay are given as association constants (Ka) in table 2. The values given here are dissociation constants (Kd) obtained by transforming the Ka values from table 2 (Kd = 1/Ka). ychi@uky.edu J. Biol. Chem. (0021-9258) 2009 Only protein-protein interactions imex curation IBD - Inflammatory bowel disease rha-2009-3 Multiple binding modes between HNF4alpha and the LXXLL motifs of PGC-1alpha lead to full activation. in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Rha G.B., Wu G., Shoelson S.E., Chi Y.I. Ulcerative colitis - Interactions of proteins identified as having a link to ulcerative colitis ychi@uky.edu J. Biol. Chem. (0021-9258) 2009 Only protein-protein interactions imex curation IBD - Inflammatory bowel disease rha-2009-4 Multiple binding modes between HNF4alpha and the LXXLL motifs of PGC-1alpha lead to full activation. in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column weight by comassie molecular weight estimation by coomasie staining weight by comassie Rha G.B., Wu G., Shoelson S.E., Chi Y.I. Ulcerative colitis - Interactions of proteins identified as having a link to ulcerative colitis ychi@uky.edu J. Biol. Chem. (0021-9258) 2009 Only protein-protein interactions imex curation IBD - Inflammatory bowel disease prgc1_human Peroxisome proliferator-activated receptor gamma coactivator 1-alpha Ligand effect modulator 6 PPARGC1A LEM6 PGC1 PGC1A PPARGC1 protein protein human Homo sapiens Human MAWDMCNQDSESVWSDIECAALVGEDQPLCPDLPELDLSELDVNDLDTDSFLGGLKWCSDQSEIISNQYNNEPSNIFEKIDEENEANLLAVLTETLDSLPVDEDGLPSFDALTDGDVTTDNEASPSSMPDGTPPPQEAEEPSLLKKLLLAPANTQLSYNECSGLSTQNHANHNHRIRTNPAIVKTENSWSNKAKSICQQQKPQRRPCSELLKYLTTNDDPPHTKPTENRNSSRDKCTSKKKSHTQSQSQHLQAKPTTLSLPLTPESPNDPKGSPFENKTIERTLSVELSGTAGLTPPTTPPHKANQDNPFRASPKLKSSCKTVVPPPSKKPRYSESSGTQGNNSTKKGPEQSELYAQLSKSSVLTGGHEERKTKRPSLRLFGDHDYCQSINSKTEILINISQELQDSRQLENKDVSSDWQGQICSSTDSDQCYLRETLEASKQVSPCSTRKQLQDQEIRAELNKHFGHPSQAVFDDEADKTGELRDSDFSNEQFSKLPMFINSGLAMDGLFDDSEDESDKLSYPWDGTQSYSLFNVSPSCSSFNSPCRDSVSPPKSLFSQRPQRMRSRSRSFSRHRSCSRSPYSRSRSRSPGSRSSSRSCYYYESSHYRHRTHRNSPLYVRSRSRSPYSRRPRYDSYEEYQHERLKREEYRREYEKRESERAKQRERQRQKAIEERRVIYVGKIRPDTTRTELRDRFEVFGEIEECTVNLRDDGDSYGFITYRYTCDAFAALENGYTLRRSNETDFELYFCGRKQFFKSNYADLDSNSDDFDPASTKSKYDSLDFDSLLKEAQRSLRR hnf4a_human Hepatocyte nuclear factor 4-alpha Transcription factor HNF-4 Nuclear receptor subfamily 2 group A member 1 Transcription factor 14 HNF4A HNF4 NR2A1 TCF14 protein protein human Homo sapiens Human MRLSKTLVDMDMADYSAALDPAYTTLEFENVQVLTMGNDTSPSEGTNLNAPNSLGVSALCAICGDRATGKHYGASSCDGCKGFFRRSVRKNHMYSCRFSRQCVVDKDKRNQCRYCRLKKCFRAGMKKEAVQNERDRISTRRSSYEDSSLPSINALLQAEVLSRQITSPVSGINGDIRAKKIASIADVCESMKEQLLVLVEWAKYIPAFCELPLDDQVALLRAHAGEHLLLGATKRSMVFKDVLLLGNDYIVPRHCPELAEMSRVSIRILDELVLPFQELQIDDNEYAYLKAIIFFDPDAKGLSDPGKIKRLRSQVQVSLEDYINDRQYDSRGRFGELLLLLPTLQSITWQMIEQIQFIKLFGMAKIDNLLQEMLLGGSPSDAPHAHHPLHPHLMQEHMGTNVIVANTMPTHLSNGQMCEWPRPRGQAATPETPQPSPPGGSGSEPYKLLPGAVATIVKPLSAIPQPTITKQEVI pparg_human Peroxisome proliferator-activated receptor gamma Nuclear receptor subfamily 1 group C member 3 PPARG NR1C3 protein protein human Homo sapiens Human MGETLGDSPIDPESDSFTDTLSANISQEMTMVDTEMPFWPTNFGISSVDLSVMEDHSHSFDIKPFTTVDFSSISTPHYEDIPFTRTDPVVADYKYDLKLQEYQSAIKVEPASPPYYSEKTQLYNKPHEEPSNSLMAIECRVCGDKASGFHYGVHACEGCKGFFRRTIRLKLIYDRCDLNCRIHKKSRNKCQYCRFQKCLAVGMSHNAIRFGRMPQAEKEKLLAEISSDIDQLNPESADLRALAKHLYDSYIKSFPLTKAKARAILTGKTTDKSPFVIYDMNSLMMGEDKIKFKHITPLQEQSKEVAIRIFQGCQFRSVEAVQEITEYAKSIPGFVNLDLNDQVTLLKYGVHEIIYTMLASLMNKDGVLISEGQGFMTREFLKSLRKPFGDFMEPKFEFAVKFNALELDDSDLAIFIAVIILSGDRPGLLNVKPIEDIQDNLLQALELQLKLNHPESSQLFAKLLQKMTDLRQIVTEHVQLLQVIKKTETDMSLHPLLQEIYKDLY hnf4a-ppargc1a 1573826 n/a 1573830 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false Q9UBK2:p.Leu147_Leu148delinsAlaAla mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false Q9UBK2:p.Tyr213_Leu214delinsAlaAla mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false Q9UBK2:p.[Val91_Leu92delinsAlaAla;Leu147_Leu148delinsAlaAla] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Q9UBK2:p.[Val91_Leu92delinsAlaAla;Tyr213_Leu214delinsAlaAla] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Q9UBK2:p.[Leu147_Leu148delinsAlaAla;Tyr213_Leu214delinsAlaAla] mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Q9UBK2:p.[Val91_Leu92delinsAlaAla;Leu147_Leu148delinsAlaAla;Tyr213_Leu214delinsAlaAla] mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis 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 in vitro In vitro n/a 1573831 unspecified role unspecified role bait bait 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 false nucl_hormone_rcpt_ligand-bd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecobd Escherichia coli (strain B / BL21-DE3) direct interaction direct interaction false false false Fig. 5a Q9UBK2 increases activity of P41235 pparg-ppargc1a-2 1573826 n/a 1573832 unspecified role unspecified role bait bait 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 false nucl_hormone_rcpt_ligand-bd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecobd Escherichia coli (strain B / BL21-DE3) n/a 1573830 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false Q9UBK2:p.Leu147_Leu148delinsAlaAla mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false Q9UBK2:p.[Val91_Leu92delinsAlaAla;Leu147_Leu148delinsAlaAla] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Q9UBK2:p.[Leu147_Leu148delinsAlaAla;Tyr213_Leu214delinsAlaAla] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Q9UBK2:p.[Val91_Leu92delinsAlaAla;Leu147_Leu148delinsAlaAla;Tyr213_Leu214delinsAlaAla] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis 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 in vitro In vitro direct interaction direct interaction false false false Suppl. Fig. S5b Triglitazone (1 micromolar) was added as an agonist for PPAR-gamma. Q9UBK2 increases activity of P37231 hnf4a-ppargc1a-1 1573827 n/a 1573830 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 false Q9UBK2:p.Val91_Leu92delinsAlaAla mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false Q9UBK2:p.Leu147_Leu148delinsAlaAla mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false Q9UBK2:p.Tyr213_Leu214delinsAlaAla mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false Q9UBK2:p.[Val91_Leu92delinsAlaAla;Leu147_Leu148delinsAlaAla] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Q9UBK2:p.[Val91_Leu92delinsAlaAla;Tyr213_Leu214delinsAlaAla] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Q9UBK2:p.[Leu147_Leu148delinsAlaAla;Tyr213_Leu214delinsAlaAla] mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Q9UBK2:p.[Val91_Leu92delinsAlaAla;Leu147_Leu148delinsAlaAla;Tyr213_Leu214delinsAlaAla] mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis 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 ecobd Escherichia coli (strain B / BL21-DE3) Stoichiometry: 1.0 n/a 1573831 unspecified role unspecified role neutral component neutral component nucl_hormone_rcpt_ligand-bd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecobd Escherichia coli (strain B / BL21-DE3) Stoichiometry: 1.0 1573866 1573857 direct interaction direct interaction false false false 1573827 Fig. 5b, Table 2 Temp = 20C, pH = 6 The wild type PGC-1alpha construct (residues 74-219) binds with Kd = 1.23 x 10E-6 molar. Mutations in the different LxxLL motifs decrease binding to: Kd = 1.95 x 10E-6 molar (val91ala_leu92ala); Kd = 1.47 x 10E-5 molar (leu147ala_leu148ala); Kd = 2.32 x 10E-6 molar (tyr213ala_leu214ala); Kd = 1.82 x 10E-5 molar (val91ala_leu92ala_leu147ala_leu148ala); and Kd = 3.00 x 10E-6 molar (val91ala_leu92ala_tyr213ala_leu214ala). hnf4a-ppargc1a-2 1573828 n/a 1573830 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 false ecobd Escherichia coli (strain B / BL21-DE3) Stoichiometry: 1.0 n/a 1573831 unspecified role unspecified role neutral component neutral component nucl_hormone_rcpt_ligand-bd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecobd Escherichia coli (strain B / BL21-DE3) Stoichiometry: 1.0 1573871 1573869 direct interaction direct interaction false false false Table 1 2.20A working 19.0%, free 23.6% Although the PGC-1alpha construct contains three LxxLL motifs, which mediate binding to the ligand-binding domain (LBD) of nuclear receptors like HNF4alpha, the crystal structure shows only one LxxLL motif bound to HNF4alpha. However, the electron densities for the side chains of the bound motif are poorly defined except for except for the leucine residues that constitute the only common residues between the three motifs in PGC-1alpha. This indicates that the bound motif in the crystal structure is not a single sequence but an averaged structure of more than one LxxLL motif. hnf4a-ppargc1a-3 1573829 n/a 1573830 unspecified role unspecified role neutral component neutral component region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false ecobd Escherichia coli (strain B / BL21-DE3) n/a 1573831 unspecified role unspecified role neutral component neutral component nucl_hormone_rcpt_ligand-bd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecobd Escherichia coli (strain B / BL21-DE3) direct interaction direct interaction false false false Suppl. Fig. S1