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/ scott-2014-1 Structure of a RING E3 trapped in action reveals ligation mechanism for the ubiquitin-like protein NEDD8. in vitro In vitro neddylase assay neddylase assay tag fluorescence tag visualisation by fluorescence tag fluorescence Scott D.C., Sviderskiy V.O., Monda J.K., Lydeard J.R., Cho S.E., Harper J.W., Schulman B.A. Pulse-chase assays were used to monitoring the ligation reaction Indicated E2-Ubiquitin-like protein (UBL) complexes were formed in the pulse reaction. After quenching the pulse, UBL was “chased” from E2 by adding additional proteins or protein complexes. Details of some constructs (e.g. tags, length and expression system) are not clear; when in doubt these have been matched to constructs used in the crystal. brenda.schulman@stjude.org Cell (0092-8674) 2014 Only protein-protein interactions imex curation scott-2014-2 Structure of a RING E3 trapped in action reveals ligation mechanism for the ubiquitin-like protein NEDD8. in vitro In vitro ubiquitinase assay ubiquitinase assay ubiquitinase assay tag fluorescence tag visualisation by fluorescence tag fluorescence Scott D.C., Sviderskiy V.O., Monda J.K., Lydeard J.R., Cho S.E., Harper J.W., Schulman B.A. Pulse-chase assays were used to monitoring the ligation reaction Indicated E2-Ubiquitin-like protein (UBL) complexes were formed in the pulse reaction. After quenching the pulse, UBL was “chased” from E2 by adding additional proteins or protein complexes. Details of some constructs (e.g. tags, length and expression system) are not clear; when in doubt these have been matched to constructs used in the crystal. brenda.schulman@stjude.org Cell (0092-8674) 2014 Only protein-protein interactions imex curation scott-2014-3 Structure of a RING E3 trapped in action reveals ligation mechanism for the ubiquitin-like protein NEDD8. in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Scott D.C., Sviderskiy V.O., Monda J.K., Lydeard J.R., Cho S.E., Harper J.W., Schulman B.A. It is not clear if the GST tags on Cullin1 and Nedd8 have been retained, or not. Details of some constructs (e.g. tags, length and expression system) are not clear; when in doubt these have been matched to constructs used in the crystal. brenda.schulman@stjude.org Cell (0092-8674) 2014 Only protein-protein interactions imex curation rbx1_human E3 ubiquitin-protein ligase RBX1 RBX1 RNF75 ROC1 Regulator of cullins 1 RING finger protein 75 Protein ZYP RING-box protein 1 E3 ubiquitin-protein transferase RBX1 protein protein human Homo sapiens Human MAAAMDVDTPSGTNSGAGKKRFEVKKWNAVALWAWDIVVDNCAICRNHIMDLCIECQANQASATSEECTVAWGVCNHAFHFHCISRWLKTRQVCPLDNREWEFQKYGH ubc12_human NEDD8-conjugating enzyme Ubc12 UBE2M UBC12 Ubiquitin-conjugating enzyme E2 M NEDD8 carrier protein protein protein human Homo sapiens Human MIKLFSLKQQKKEEESAGGTKGSSKKASAAQLRIQKDINELNLPKTCDISFSDPDDLLNFKLVICPDEGFYKSGKFVFSFKVGQGYPHDPPKVKCETMVYHPNIDLEGNVCLNILREDWKPVLTINSIIYGLQYLFLEPNPEDPLNKEAAEVLQNNRRLFEQNVQRSMRGGYIGSTYFERCLK cul1_human Cullin-1 CUL1 protein protein human Homo sapiens Human MSSTRSQNPHGLKQIGLDQIWDDLRAGIQQVYTRQSMAKSRYMELYTHVYNYCTSVHQSNQARGAGVPPSKSKKGQTPGGAQFVGLELYKRLKEFLKNYLTNLLKDGEDLMDESVLKFYTQQWEDYRFSSKVLNGICAYLNRHWVRRECDEGRKGIYEIYSLALVTWRDCLFRPLNKQVTNAVLKLIEKERNGETINTRLISGVVQSYVELGLNEDDAFAKGPTLTVYKESFESQFLADTERFYTRESTEFLQQNPVTEYMKKAEARLLEEQRRVQVYLHESTQDELARKCEQVLIEKHLEIFHTEFQNLLDADKNEDLGRMYNLVSRIQDGLGELKKLLETHIHNQGLAAIEKCGEAALNDPKMYVQTVLDVHKKYNALVMSAFNNDAGFVAALDKACGRFINNNAVTKMAQSSSKSPELLARYCDSLLKKSSKNPEEAELEDTLNQVMVVFKYIEDKDVFQKFYAKMLAKRLVHQNSASDDAEASMISKLKQACGFEYTSKLQRMFQDIGVSKDLNEQFKKHLTNSEPLDLDFSIQVLSSGSWPFQQSCTFALPSELERSYQRFTAFYASRHSGRKLTWLYQLSKGELVTNCFKNRYTLQASTFQMAILLQYNTEDAYTVQQLTDSTQIKMDILAQVLQILLKSKLLVLEDENANVDEVELKPDTLIKLYLGYKNKKLRVNINVPMKTEQKQEQETTHKNIEEDRKLLIQAAIVRIMKMRKVLKHQQLLGEVLTQLSSRFKPRVPVIKKCIDILIEKEYLERVDGEKDTYSYLA nedd8_human NEDD8 Ubiquitin-like protein Nedd8 Neddylin Neural precursor cell expressed developmentally down-regulated protein 8 NEDD8 protein protein human Homo sapiens Human MLIKVKTLTGKEIEIDIEPTDKVERIKERVEEKEGIPPQQQRLIYSGKQMNDEKTAADYKILGGSVLHLVLALRGGGGLRQ ub2d2_human Ubiquitin-conjugating enzyme E2 D2 UBC5B UBCH4 UBE2D2 UBC4 UBCH5B Ubiquitin-protein ligase D2 Ubiquitin carrier protein D2 Ubiquitin-conjugating enzyme E2-17 kDa 2 Ubiquitin-conjugating enzyme E2(17)KB 2 PUBC1 p53-regulated ubiquitin-conjugating enzyme 1 (E3-independent) E2 ubiquitin-conjugating enzyme D2 E2 ubiquitin-conjugating enzyme D2 protein protein human Homo sapiens Human MALKRIHKELNDLARDPPAQCSAGPVGDDMFHWQATIMGPNDSPYQGGVFFLTIHFPTDYPFKPPKVAFTTRIYHPNINSNGSICLDILRSQWSPALTISKVLLSICSLLCDPNPDDPLVPEIARIYKTDREKYNRIAREWTQKYAM ubiq_human UBIQ_HUMAN protein protein human Homo sapiens Human MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG dcnl1_human DCN1-like protein 1 DCUN1D1 DCUN1L1 RP42 SCCRO Defective in cullin neddylation protein 1-like protein 1 DCUN1 domain-containing protein 1 Squamous cell carcinoma-related oncogene DCN1 protein protein human Homo sapiens Human MNKLKSSQKDKVRQFMIFTQSSEKTAVSCLSQNDWKLDVATDNFFQNPELYIRESVKGSLDRKKLEQLYNRYKDPQDENKIGIDGIQQFCDDLALDPASISVLIIAWKFRAATQCEFSKQEFMDGMTELGCDSIEKLKAQIPKMEQELKEPGRFKDFYQFTFNFAKNPGQKGLDLEMAIAYWNLVLNGRFKFLDLWNKFLLEHHKRSIPKDTWNLLLDFSTMIADDMSNYDEEGAWPVLIDDFVEFARPQIAGTKSTTV cul1-nedd8-1 1566661 n/a 1566664 enzyme enzyme 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 P62877:p.Arg46Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false P62877:p.Asn98Arg mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false P62877:p.Ile37Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false P62877:p.Ile37_Val38delinsGlyGly mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false P62877:p.Trp35Asp 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) n/a 1566665 ancillary ancillary neutral component neutral component P61081:p.Asn113Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false 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 P61081:p.Arg116Ala 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) n/a 1566666 enzyme target enzyme target substrate substrate neutral component neutral component c-terminal region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Q13616:p.[Leu421Glu;Val451_Val452delinsGluLys;Tyr455Lys] mutation mutation 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 Q13616:p.Lys720Ala mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1566667 ancillary ancillary neutral component neutral component 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region fluorescent dye fluorescent dye label fluorescent dye undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false Q15843:p.Ile44Ala mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain false Q15843:p.Leu8Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false Q15843:p.Leu8_Thr9delinsAlaAla mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain false Q15843:p.Glu32Asp mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false Q15843:p.Glu31_Glu32delinsGlnAsp mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) neddylation neddylation reaction neddylation false false false Fig. 1B, 1D, 1G, 3D, 3H, 3K, 3L, 4D, 4E, 4G. Supp. Fig. 1C, 1D, Subsequent analysis of the product by western blot confirmed the fluorescent tag on NEDD8 had no affect on reaction rate. Introduction of lysines surrounding the CUL1 lys720ala substitution, or appending Gly-Gly-Lys to the C terminus fails to rescue CUL1 neddylation. UBC12 tyr130leu mutations rescues NEDD8 ile44ala mutation. cul1-rbx1-11 1566661 n/a 1566664 enzyme enzyme 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 P62877:p.Asn98Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false P62877:p.Asn98Arg mutation increasing rate mutation increasing interaction rate mutation increasing rate certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1566667 ancillary ancillary neutral component neutral component region fluorescent dye fluorescent dye label fluorescent dye undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false Q15843:p.Ala72Arg mutation increasing rate mutation increasing interaction rate mutation increasing rate 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 ecoli Escherichia coli (strain K12) n/a 1566668 ancillary ancillary neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1566666 enzyme target enzyme target substrate substrate neutral component neutral component Q13616:p.[Leu421Glu;Val451_Val452delinsGluLys;Tyr455Lys] mutation mutation 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 c-terminal region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) neddylation neddylation reaction neddylation false false false Fig. 1E RBX1 promotingted ubiquitin ligation from UBCH5 to CUL1 more slowly that is did ligation from UBC12-NEDD8. cul1-rbx1-9 1566662 n/a 1566669 ancillary ancillary neutral component neutral component region fluorescent dye fluorescent dye label fluorescent dye undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ile44ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false asp32glu mutation increasing rate mutation increasing interaction rate mutation increasing rate certain certain sequence position certain certain certain sequence position certain false gln31glu,asp32glu mutation increasing rate mutation increasing interaction rate mutation increasing rate certain certain sequence position certain certain certain sequence position certain false n/a 1566668 ancillary ancillary neutral component neutral component P62837:p.Ser108Leu mutation increasing rate mutation increasing interaction rate mutation increasing rate certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1566664 enzyme enzyme 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 P62877:p.Asn98Arg mutation increasing rate mutation increasing interaction rate mutation increasing rate certain certain sequence position certain certain certain sequence position certain false P62877:p.Asn98Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false P62877:p.Trp35Asp 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) n/a 1566666 enzyme target enzyme target substrate substrate neutral component neutral component Q13616:p.[Leu421Glu;Val451_Val452delinsGluLys;Tyr455Lys] mutation mutation 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 c-terminal region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) ubiquitination ubiquitination reaction ubiquitination false false false Fig. 1C, 3E, 4F, 4H RBX1 promotingted ubiquitin ligation from UBCH5 to CUL1 more slowly that is did ligation from UBC12-NEDD8. UBCH5B ser108leu mutations rescue UBIQ ile44ala mutations cul1-rbx1-10 1566662 n/a 1566664 enzyme enzyme neutral component neutral component P62877:p.Asn98Arg mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate 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 ecoli Escherichia coli (strain K12) n/a 1566669 ancillary ancillary neutral component neutral component arg72ala mutation increasing rate mutation increasing interaction rate mutation increasing rate certain certain sequence position certain certain certain sequence position certain false n/a 1566665 ancillary ancillary neutral component neutral component 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 ecoli Escherichia coli (strain K12) n/a 1566666 enzyme target enzyme target substrate substrate neutral component neutral component Q13616:p.[Leu421Glu;Val451_Val452delinsGluLys;Tyr455Lys] mutation mutation 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 c-terminal region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) ubiquitination ubiquitination reaction ubiquitination false false false Fig. 1D cul1-dcun1d1 1566663 n/a 1566670 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 ecoli Escherichia coli (strain K12) n/a 1566664 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 ecoli Escherichia coli (strain K12) n/a 1566665 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 P61081:p.[Asn103Ser;Cys111Ser] mutation mutation certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. n/a 1566667 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 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 ecoli Escherichia coli (strain K12) n/a 1566666 unspecified role unspecified role neutral component neutral component Q13616:p.[Leu421Glu;Val451_Val452delinsGluLys;Tyr455Lys;Lys720Arg] mutation mutation 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 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 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 ecoli Escherichia coli (strain K12) physical association physical association false false false 3.1A Rworking = 0.23 Rfree = 0.28 Supp. Table 1B