DIP Database of Interacting Proteins 611 Young Drive East; Los Angeles CA 90095; USA http://dip.doe-mbi.ucla.edu/ dip@mbi.ucla.edu 611 Young Drive East; Los Angeles CA 90095; USA http://dip.doe-mbi.ucla.edu/ levin-2010-2 in vitro In vitro ubiquitinase assay ubiquitinase assay ubiquitinase assay western blot western blot Immuno blot Immuno blot Levin I., Eakin C., Blanc MP., Klevit RE., Miller SI., Brzovic PS., Levin I., Eakin C., Blanc MP., Klevit RE., Miller SI., Brzovic PS., Levin I., Eakin C., Blanc MP., Klevit RE., Miller SI., Brzovic PS., Levin I., Eakin C., Blanc MP., Klevit RE., Miller SI., Brzovic PS. In Fig S2 a His-tag is stated to be present on Ssph2 OR E1 SspH2-catalyzed reactions were performed with either purified UbcH5 ∼ Ub conjugates or in reaction mixtures containing E1 and ubiquitin (origins unknown). klevit@u.washington.edu Proceedings of the National Academy of Sciences of the United States of America 2010 imex curation Only protein-protein interactions levin-2010-1 in vitro In vitro nmr nuclear magnetic resonance NMR NMR nmr predetermined predetermined participant predetermined Levin I., Eakin C., Blanc MP., Klevit RE., Miller SI., Brzovic PS., Levin I., Eakin C., Blanc MP., Klevit RE., Miller SI., Brzovic PS., Levin I., Eakin C., Blanc MP., Klevit RE., Miller SI., Brzovic PS., Levin I., Eakin C., Blanc MP., Klevit RE., Miller SI., Brzovic PS. klevit@u.washington.edu Proceedings of the National Academy of Sciences of the United States of America 2010 imex curation Only protein-protein interactions levin-2010-3 in vitro In vitro ubiquitinase assay ubiquitinase assay ubiquitinase assay weight by comassie molecular weight estimation by coomasie staining weight by comassie Levin I., Eakin C., Blanc MP., Klevit RE., Miller SI., Brzovic PS., Levin I., Eakin C., Blanc MP., Klevit RE., Miller SI., Brzovic PS., Levin I., Eakin C., Blanc MP., Klevit RE., Miller SI., Brzovic PS., Levin I., Eakin C., Blanc MP., Klevit RE., Miller SI., Brzovic PS. SspH2-catalyzed reactions were performed with either purified UbcH5 ∼ Ub conjugates or in reaction mixtures containing E1 and ubiquitin (origins unknown). klevit@u.washington.edu Proceedings of the National Academy of Sciences of the United States of America 2010 imex curation Only protein-protein interactions levin-2010-4 in vitro In vitro ubiquitinase assay ubiquitinase assay ubiquitinase assay ms participant Identification by mass spectrometry ms participant Levin I., Eakin C., Blanc MP., Klevit RE., Miller SI., Brzovic PS., Levin I., Eakin C., Blanc MP., Klevit RE., Miller SI., Brzovic PS., Levin I., Eakin C., Blanc MP., Klevit RE., Miller SI., Brzovic PS., Levin I., Eakin C., Blanc MP., Klevit RE., Miller SI., Brzovic PS. SspH2-catalyzed reactions were performed with either purified UbcH5 ∼ Ub conjugates or in reaction mixtures containing E1 and ubiquitin (origins unknown). klevit@u.washington.edu Proceedings of the National Academy of Sciences of the United States of America 2010 imex curation Only protein-protein interactions ssph2_salty E3 ubiquitin-protein ligase SspH2 Salmonella secreted protein H2 Secreted effector protein sspH2 STM2241 sspH2 RING-type E3 ubiquitin transferase SspH2 protein protein salty Salmonella typhimurium MPFHIGSGCLPATISNRRIYRIAWSDTPPEMSSWEKMKEFFCSTHQTEALECIWTICHPPAGTTREDVINRFELLRTLAYAGWEESIHSGQHGENYFCILDEDSQEILSVTLDDAGNYTVNCQGYSETHRLTLDTAQGEEGTGHAEGASGTFRTSFLPATTAPQTPAEYDAVWSAWRRAAPAEESRGRAAVVQKMRACLNNGNAVLNVGESGLTTLPDCLPAHITTLVIPDNNLTSLPALPPELRTLEVSGNQLTSLPVLPPGLLELSIFSNPLTHLPALPSGLCKLWIFGNQLTSLPVLPPGLQELSVSDNQLASLPALPSELCKLWAYNNQLTSLPMLPSGLQELSVSDNQLASLPTLPSELYKLWAYNNRLTSLPALPSGLKELIVSGNRLTSLPVLPSELKELMVSGNRLTSLPMLPSGLLSLSVYRNQLTRLPESLIHLSSETTVNLEGNPLSERTLQALREITSAPGYSGPIIRFDMAGASAPRETRALHLAAADWLVPAREGEPAPADRWHMFGQEDNADAFSLFLDRLSETENFIKDAGFKAQISSWLAQLAEDEALRANTFAMATEATSSCEDRVTFFLHQMKNVQLVHNAEKGQYDNDLAALVATGREMFRLGKLEQIAREKVRTLALVDEIEVWLAYQNKLKKSLGLTSVTSEMRFFDVSGVTVTDLQDAELQVKAAEKSEFREWILQWGPLHRVLERKAPERVNALREKQISDYEETYRMLSDTELRPSGLVGNTDAERTIGARAMESAKKTFLDGLRPLVEEMLGSYLNVQWRRN ub2d3_human Ubiquitin-conjugating enzyme E2 D3 UBC5C Ubiquitin-protein ligase D3 Ubiquitin carrier protein D3 Ubiquitin-conjugating enzyme E2-17 kDa 3 Ubiquitin-conjugating enzyme E2(17)KB 3 UBE2D3 UBCH5C (E3-independent) E2 ubiquitin-conjugating enzyme D3 E2 ubiquitin-conjugating enzyme D3 protein protein human Homo sapiens Human MALKRINKELSDLARDPPAQCSAGPVGDDMFHWQATIMGPNDSPYQGGVFFLTIHFPTDYPFKPPKVAFTTRIYHPNINSNGSICLDILRSQWSPALTISKVLLSICSLLCDPNPDDPLVPEIARIYKTDRDKYNRISREWTQKYAM ssph1_salt1 E3 ubiquitin-protein ligase SspH1 sspH1 Salmonella secreted protein H1 Secreted effector protein SspH1 STM14_1483 RING-type E3 ubiquitin transferase SspH1 protein protein salt1 Salmonella typhimurium (strain 14028s / SGSC 2262) MFNIRNTQPSVSMQAIAGAAAPEASPEEIVWEKIQVFFPQENYEEAQQCLAELCHPARGMLPDHISSQFARLKALTFPAWEENIQCNRDGINQFCILDAGSKEILSITLDDAGNYTVNCQGYSEAHDFIMDTEPGEECTEFAEGASGTSLRPATTVSQKAAEYDAVWSKWERDAPAGESPGRAAVVQEMRDCLNNGNPVLNVGASGLTTLPDRLPPHITTLVIPDNNLTSLPELPEGLRELEVSGNLQLTSLPSLPQGLQKLWAYNNWLASLPTLPPGLGDLAVSNNQLTSLPEMPPALRELRVSGNNLTSLPALPSGLQKLWAYNNRLTSLPEMSPGLQELDVSHNQLTRLPQSLTGLSSAARVYLDGNPLSVRTLQALRDIIGHSGIRIHFDMAGPSVPREARALHLAVADWLTSAREGEAAQADRWQAFGLEDNAAAFSLVLDRLRETENFKKDAGFKAQISSWLTQLAEDAALRAKTFAMATEATSTCEDRVTHALHQMNNVQLVHNAEKGEYDNNLQGLVSTGREMFRLATLEQIAREKAGTLALVDDVEVYLAFQNKLKESLELTSVTSEMRFFDVSGVTVSDLQAAELQVKTAENSGFSKWILQWGPLHSVLERKVPERFNALREKQISDYEDTYRKLYDEVLKSSGLVDDTDAERTIGVSAMDSAKKEFLDGLRALVDEVLGSYLTARWRLN ssph2-ube2d3-2 1792172 N/A 1792176 unspecified role unspecified role neutral component neutral component region 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false N/A 1792177 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) ubiquitination ubiquitination reaction ubiquitination false false false S1 and S2 In Fig S2 a His-tag is stated to be present on Ssph2 OR E1 ssph2-ube2d3-4 1792172 N/A 1792176 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 N/A 1792177 unspecified role unspecified role neutral component neutral component P61077:p.Cys85Ser mutation mutation certain certain sequence position certain certain certain sequence position certain false cys85 oglycoser O-glycosyl-L-serine OGlycoSer certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) ubiquitination ubiquitination reaction ubiquitination false false false 5 The UbcH5 molecule has an active site C85S mutation, and through this site is linked to Ubiquitin via an ester link, rather than through a thio-ester link. This UbcH5-O-Ub conjugate is stable. ssph2-ube2d3-3 1792172 N/A 1792176 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 N/A 1792177 unspecified role unspecified role neutral component neutral component P61077:p.Ala96Asp mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false P61077:p.Phe62Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) ubiquitination ubiquitination reaction ubiquitination false false false 1B, 4, S6 and 3B ssph1-ube2d3-1 1792172 N/A 1792178 unspecified role unspecified role neutral component neutral component N/A 1792177 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) ubiquitination ubiquitination reaction ubiquitination false false false S2 ssph2-ube2d3-7 1792172 N/A 1792176 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 P0CE12:p.Cys580Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false N/A 1792177 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) ubiquitination ubiquitination reaction ubiquitination false false false S2,6 ssph2-ubch5-1 1792173 N/A 1792176 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 N/A 1792177 unspecified role unspecified role neutral component neutral component cys85 oglycoser O-glycosyl-L-serine OGlycoSer certain certain sequence position certain certain certain sequence position certain false P61077:p.Cys85Ser mutation mutation certain certain sequence position certain certain certain sequence position certain false P61077:p.Ala96Asp mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false 15n label 15n label 15n label 15N N15 15N N15 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecoli Escherichia coli (strain K12) physical association physical association false false false 2 and 3A SspH2 preferentially binds the substrate (i.e., UbcH5 ∼ Ub) as opposed to the product (i.e., free UbcH5) of the Ub-transfer reaction. The UbcH5 molecule has an active site C85S mutation, and through this site is linked to Ubiquitin via an ester link, rather than through a thio-ester link. This UbcH5-O-Ub conjugate is stable. ssph2-ube2d3-5 1792174 N/A 1792176 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 N/A 1792177 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) ubiquitination ubiquitination reaction ubiquitination false false false S3A The use of K48R-Ub generates high MW ubiquitylation products ssph2-ube2d3-6 1792175 N/A 1792176 unspecified role unspecified role neutral component neutral component N/A 1792177 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) ubiquitination ubiquitination reaction ubiquitination false false false S3B