MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Structure of human Sp140 PHD finger: an atypical fold interacting with Pin1 Structure of human Sp140 PHD finger: an atypical fold interacting with Pin1 FEBS J. (1742-464X) 2013 imex curation Zucchelli C., Tamburri S., Quilici G., Palagano E., Berardi A., Saare M., Peterson P., Bachi A., Musco G. musco.giovanna@hsr.it 13-Jan-2014: Contacted by IntAct-Help. Only protein-protein interactions human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Zucchelli C., Tamburri S., Quilici G., Palagano E., Berardi A., Saare M., Peterson P., Bachi A., Musco G. musco.giovanna@hsr.it Accepted 2013-OCT-28 by LUANA FEBS J. (1742-464X) 2013 Only protein-protein interactions imex curation Structure of human Sp140 PHD finger: an atypical fold interacting with Pin1 Structure of human Sp140 PHD finger: an atypical fold interacting with Pin1 FEBS J. (1742-464X) 2013 imex curation Zucchelli C., Tamburri S., Quilici G., Palagano E., Berardi A., Saare M., Peterson P., Bachi A., Musco G. musco.giovanna@hsr.it 13-Jan-2014: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro nmr nuclear magnetic resonance NMR NMR nmr predetermined predetermined participant predetermined Zucchelli C., Tamburri S., Quilici G., Palagano E., Berardi A., Saare M., Peterson P., Bachi A., Musco G. musco.giovanna@hsr.it Accepted 2013-OCT-28 by LUANA FEBS J. (1742-464X) 2013 Only protein-protein interactions imex curation pin1_human Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 Rotamase Pin1 Peptidyl-prolyl cis-trans isomerase Pin1 PIN1 protein protein human Homo sapiens Human MADEEKLPPGWEKRMSRSSGRVYYFNHITNASQWERPSGNSSSGGKNGQGEPARVRCSHLLVKHSQSRRPSSWRQEKITRTKEEALELINGYIQKIKSGEEDFESLASQFSDCSSAKARGDLGAFSRGQMQKPFEDASFALRTGEMSGPVFTDSGIHIILRTE DIP protein Q13526 original sequence version: 65 DIP protein Q13526 original sequence version: 64 DIP protein Q13526 original sequence version: 163 DIP protein Q13526 original sequence version: 156 DIP protein Q13526 original sequence version: 117 35391AF40B7D1E13 sp140_human Nuclear body protein SP140 SP140 LYSP100 Lymphoid-restricted homolog of Sp100 Nuclear autoantigen Sp-140 Speckled 140 kDa protein protein human Homo sapiens Human MAQQGQQGQMASGDSNLNFRMVAEIQNVEGQNLQEQVCPEPIFRFFRENKVEIASAITRPFPFLMGLRDRSFISEQMYEHFQEAFRNLVPVTRVMYCVLSELEKTFGWSHLEALFSRINLMAYPDLNEIYRSFQNVCYEHSPLQMNNVNDLEDRPRLLPYGKQENSNACHEMDDIAVPQEALSSSPRCEPGFSSESCEQLALPKAGGGDAEDAPSLLPGGGVSCKLAIQIDEGESEEMPKLLPYDTEVLESNGMIDAARTYSTAPGEKQGEEEGRNSPRKRNQDKEKYQESPEGRDKETFDLKTPQVTNEGEPEKGLCLLPGEGEEGSDDCSEMCDGEEPQEASSSLARCGSVSCLSAETFDLKTPQVTNEGEPEKELSLLPGEGEEGSDDCSEMCDGEERQEASSSLARRGSVSSELENHPMNEEGESEELASSLLYDNVPGAEQSAYENEKCSCVMCFSEEVPGSPEARTESDQACGTMDTVDIANNSTLGKPKRKRRKKRGHGWSRMRMRRQENSQQNDNSKADGQVVSSEKKANVNLKDLSKIRGRKRGKPGTRFTQSDRAAQKRVRSRASRKHKDETVDFKAPLLPVTCGGVKGILHKKKLQQGILVKCIQTEDGKWFTPTEFEIKGGHARSKNWRLSVRCGGWPLRWLMENGFLPDPPRIRYRKKKRILKSQNNSSVDPCMRNLDECEVCRDGGELFCCDTCSRVFHEDCHIPPVEAERTPWNCIFCRMKESPGSQQCCQESEVLERQMCPEEQLKCEFLLLKVYCCSESSFFAKIPYYYYIREACQGLKEPMWLDKIKKRLNEHGYPQVEGFVQDMRLIFQNHRASYKYKDFGQMGFRLEAEFEKNFKEVFAIQETNGNN 355601D1D4689A74 pin1-sp140 2 Pin1 3 predetermined predetermined participant predetermined predetermined predetermined participant predetermined enzyme enzyme unspecified role unspecified role purified purified his tag 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 Sp140-PHD L3 loop 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined enzyme target enzyme target substrate substrate unspecified role unspecified role in vitro translated in vitro translated protein in vitro translated pthr726 phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) predetermined featur predetermined feature predetermined featur certain certain sequence position certain certain certain sequence position certain binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain proline isomerization reaction proline isomerization reaction f2a To this aim we first tested in vitro Pin1 enzyme activity on two peptides, EAERpTPWN and EAERTPWN, corresponding to Sp140-PHD L3 loop (Glu722-Asn729), with or without Threonine phosphorylation, respectively. Because of the slow exchange rate of the peptidyl prolyl cis-trans isomerization, several residues in both the free peptides displayed two distinct sets of 1H signals in 2D ROESY experiments (Fig. 2A-B). The cis and trans populations of both peptides were 15% and 85%, respectively, as estimated from the one-dimensional 1H and 2D 1H-13C HSQC spectra at room temperature. Exchange cross peaks were absent in the ROESY spectra of the free peptides, indicating that the exchange regime between the two conformations was too slow to be detected on the NMR timescale (Fig. 2A left, 2B left). Notably, addition of catalytic amounts of Pin1 to EAERpTPWN accelerated the isomerization rate of the phosphothreonine-prolyl bond, as shown by the appearance of exchange cross-peaks in the ROESY spectrum (Fig. 2A, right). As expected, in the presence of Pin1 no exchange peaks were observed for the non-phosphorylated control peptide (Fig. 2B, right). pin1-sp140-1 2 Pin1 3 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role unspecified role unspecified role purified purified his tag 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 Sp140-PHD L3 loop 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role unspecified role unspecified role in vitro translated in vitro translated protein in vitro translated binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain pthr726 phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) predetermined featur predetermined feature predetermined featur certain certain sequence position certain certain certain sequence position certain direct interaction direct interaction f2c f2d f2e sf8a We next determined the binding site of EAERpTPWN on Pin1, performing NMR based chemical shift mapping assays. To this end 2D 1H-15N HSQC spectra of full-length 15Nlabeled Pin1 were recorded to monitor possible changes in Pin1 1H-15N chemical shifts upon successive additions of unlabelled peptides. A comparison of the spectra in the absence and presence of a five-fold excess (1 mM) of Sp140-peptides showed that only EAERpTPWN was able to bind Pin1, as revealed by the numerous peaks' displacements observed upon addition of the phosphopeptide (Fig. 2C, Fig. S8A). The unphosphorylated peptide did not show any binding evidence, confirming the phospho-dependence of the Pin1-peptide interaction (Fig. S8B). The complex between Pin1 and EAERpTPWN was in the fast exchange regime on the NMR chemical-shift time scale (Fig. 2C) with a dissociation constant of 138 ± 4 μM (Fig. 2D). Pin1 residues exhibiting significant amide chemical shift changes (Fig. 2E) where mapped on Pin1 crystallographic structure (pdb code: 1PIN). The binding surface mainly involved the β-sheet of the WW domain (Lys13- Ser16, Gly20, Val22-Asn26, Ala31, Gln33-Arg35) (Fig. 2F). Chemical shift changes were observed also on the flexible linker (Ser41, Ser43, Lys46) and on the region of the PPIase domain facing (Lys97-Glu100) or nearby (Phe139-Arg142) the WW domain, probably induced by long range conformational rearrangements upon complex formation. Overall, these data indicate that Pin1 binds to EAERpTPWN and catalyzes the cis-trans isomerization of its phosphothreonine-proline bond in vitro. pin1-sp140-2 2 Sp140-PHD 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role unspecified role unspecified role purified purified binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain Pin1 3 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role unspecified role unspecified role purified purified his tag 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 direct interaction direct interaction f3 f4 sf9 sf10 Pin1 recognizes the Sp140-PHD scaffold independently from phosphorylation sp140-pin1 1 Sp140 4 anti tag western anti tag western blot anti tag western western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Pin1 3 western blot western blot Immuno blot Immuno blot western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f5