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/ PD-L1 and PD-L2 differ in their molecular mechanisms of interaction with PD-1. PD-L1 and PD-L2 differ in their molecular mechanisms of interaction with PD-1. International immunology 2010 imex curation Ghiotto M., Gauthier L., Serriari N., Pastor S., Truneh A., Nunès JA., Olive D. Two sources of PD-L1-Fc are used here: PD-L1-Fc recombinant protein "purchased from R&D Systems, Catalog number 156-B7" and also "produced at the laboratory". It is stated that "These two proteins "give reliable and comparable results.""but does not state when each form(s) were used. The start/end sequences of ECDs of human PD-L1/L2 were were found by primer Blat. CD80 has been assumed to be of Human origin from the statement below fig 3: ".....that CTLA-4 and PD-L1 can bind to human CD80 simultaneously.". daniel.olive@inserm.fr 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Ghiotto M., Gauthier L., Serriari N., Pastor S., Truneh A., Nunès JA., Olive D. Two sources of PD-L1-Fc are used here: PD-L1-Fc recombinant protein "purchased from R&D Systems, Catalog number 156-B7" and also "produced at the laboratory". It is stated that "These two proteins "give reliable and comparable results." but does not state when each form(s) were used. I dont know what the EXperimental Prep is meant to be if a supplier does not state it - e.g. CD80-Fc here. Two sources of PD-L1-Fc are used here: PD-L1-Fc recombinant protein "purchased from R&D Systems, Catalog number 156-B7" and also "produced at the laboratory". It is stated that "These two proteins "give reliable and comparable results.""but does not state when each form(s) were used. The start/end sequences of ECDs of human PD-L1/L2 were were found by primer Blat. CD80 has been assumed to be of Human origin from the statement below fig 3: ".....that CTLA-4 and PD-L1 can bind to human CD80 simultaneously.". daniel.olive@inserm.fr aniel.olive@inserm.fr Accepted 2018-MAY-21 AT 11:27 BST AT 11:27 BST by PPORRAS International immunology 2010 Only protein-protein interactions imex curation imex curation PD-L1 and PD-L2 differ in their molecular mechanisms of interaction with PD-1. PD-L1 and PD-L2 differ in their molecular mechanisms of interaction with PD-1. International immunology 2010 imex curation Ghiotto M., Gauthier L., Serriari N., Pastor S., Truneh A., Nunès JA., Olive D. Two sources of PD-L1-Fc are used here: PD-L1-Fc recombinant protein "purchased from R&D Systems, Catalog number 156-B7" and also "produced at the laboratory". It is stated that "These two proteins "give reliable and comparable results.""but does not state when each form(s) were used. The start/end sequences of ECDs of human PD-L1/L2 were were found by primer Blat. CD80 has been assumed to be of Human origin from the statement below fig 3: ".....that CTLA-4 and PD-L1 can bind to human CD80 simultaneously.". daniel.olive@inserm.fr 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro facs fluorescence-activated cell sorting FACS Flow cytometry facs antibody detection identification by antibody antibody detection Ghiotto M., Gauthier L., Serriari N., Pastor S., Truneh A., Nunès JA., Olive D. I have not added Fig 3C - lower image, as it is not clear what CTLA4 is binding to - Raji cells express CD86 as well as CD80 + other proteins? -no mention of transfection of CD80. Two sources of PD-L1-Fc are used here: PD-L1-Fc recombinant protein "purchased from R&D Systems, Catalog number 156-B7" and also "produced at the laboratory". It is stated that "These two proteins "give reliable and comparable results.""but does not state when each form(s) were used. The start/end sequences of ECDs of human PD-L1/L2 were were found by primer Blat. CD80 has been assumed to be of Human origin from the statement below fig 3: ".....that CTLA-4 and PD-L1 can bind to human CD80 simultaneously.". daniel.olive@inserm.fr aniel.olive@inserm.fr Accepted 2018-MAY-21 AT 11:27 BST AT 11:27 BST by PPORRAS International immunology 2010 Only protein-protein interactions imex curation imex curation pdcd1_human Programmed cell death protein 1 PDCD1 PD1 protein protein human Homo sapiens Human MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL DIP protein Q15116 original sequence version: 143 A5210FD40C304FB7 pd1l1_human Programmed cell death 1 ligand 1 CD274 B7H1 PDCD1L1 PDCD1LG1 PDL1 B7 homolog 1 protein protein human Homo sapiens Human MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET FE957086E62A31A8 pd1l2_human Programmed cell death 1 ligand 2 Butyrophilin B7-DC PDCD1LG2 B7DC CD273 PDCD1L2 PDL2 protein protein human Homo sapiens Human MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIPFCIIAFIFIATVIALRKQLCQKLYSSKDTTKRPVTTTKREVNSAI 8B7E963C9BA26ED9 cd80_human T-lymphocyte activation antigen CD80 CD80 CD28LG CD28LG1 LAB7 Activation B7-1 antigen CTLA-4 counter-receptor B7.1 BB1 protein protein human Homo sapiens Human MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV BA453EE34528B1F4 ctla4_human Cytotoxic T-lymphocyte protein 4 CTLA4 CD152 Cytotoxic T-lymphocyte-associated antigen 4 protein protein human Homo sapiens Human MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN DIP protein P16410 original sequence version: 178 6F9466FB2E139A5A pdcd1-cd274-3 1 3 unspecified role unspecified role bait bait purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos Chlorocebus aethiops cos cos 4 unspecified role unspecified role prey prey ecd sufficient to bind sufficient binding region sufficient to bind range range range range region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction Table 1, fig 1, 2, Supp fig 2/3 The conformational change model gave the best and reliable fit. Pre-incubation of PD-L1 with three different antibodies against it influenced the interaction outcome. One of the anitbodies (PD-L1.3) disrupted binding, while the ohter two (PD-L1.1 and PD-L1.2) seemed to strengthen it, probably by increasing PD-L1 stability. pdcd1-cd274-5 1 3 unspecified role unspecified role bait bait purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos Chlorocebus aethiops cos cos 5 unspecified role unspecified role prey prey purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos Chlorocebus aethiops cos cos direct interaction direct interaction Table 1, fig 1,3, Supp fig 1 pdcd1-pdcd1lg2-3 1 4 competitor competitor competitor prey prey purified purified region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecd sufficient to bind sufficient binding region sufficient to bind range range range range 3 unspecified role unspecified role bait bait ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos Chlorocebus aethiops cos cos 5 unspecified role unspecified role prey prey purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos Chlorocebus aethiops cos cos physical association physical association Fig 3A -top PD-L1 can out-compete PD-L2 following saturation of PD-L2 occupancy pdcd1-pdcd1lg2-4 1 4 unspecified role unspecified role bait bait region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecd sufficient to bind sufficient binding region sufficient to bind range range range range 3 unspecified role unspecified role prey prey purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos Chlorocebus aethiops cos cos 5 competitor competitor competitor prey prey purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos Chlorocebus aethiops cos cos physical association physical association Fig 3A -middle PD-1 proteins were pre-incubated with PD-L2 and the resulting complexes were injected onto PD-L1-Ig chips;PD-L2 pre-incubation prevented PD-1 binding to PD-L1 in a dose-dependent manner cd274-ctla4-2 2 6 unspecified role unspecified role neutral component neutral component c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 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 ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc 4 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos Chlorocebus aethiops cos cos 7 unspecified role unspecified role neutral component neutral component region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos Chlorocebus aethiops cos cos physical association physical association Fig 3C top It is not stated if the PD-L1 molecule was full length or ECD only. CTLA-4-Ig can bind to PD-L1 expressing COS cells in presence of CD80 but not PD-1. cd274-cd80-6 1 6 unspecified role unspecified role bait bait c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 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 ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc 4 unspecified role unspecified role prey prey region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecd sufficient to bind sufficient binding region sufficient to bind range range range range direct interaction direct interaction Fig 1A, 3, Table 1 the parameters of PD-L1 binding to CD80 fitted also with the conformational change model. cd274-cd80-7 1 6 unspecified role unspecified role bait bait c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 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 ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc 7 unspecified role unspecified role prey prey purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos Chlorocebus aethiops cos cos direct interaction direct interaction Fig 1A, Table 1 I cannot see any comment about Stoich for this interaction - though one would assume from curves it is 1:1 cd80-cd274-3 1 3 competitor competitor competitor prey prey purified purified region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 6 unspecified role unspecified role bait bait c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 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 ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc 4 unspecified role unspecified role prey prey region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecd sufficient to bind sufficient binding region sufficient to bind range range range range direct interaction direct interaction 3A bottom cd80-ctla4-2 1 7 unspecified role unspecified role prey prey purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos Chlorocebus aethiops cos cos 4 unspecified role unspecified role bait bait ecd sufficient to bind sufficient binding region sufficient to bind range range range range region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 6 ancillary ancillary prey prey c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 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 ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc physical association physical association Supp fig 4-2 not sure if "Biological" Role of Ancillary is OK here - for CD80? CTLA-4 cannot directly bind to PD-L1 chip but strongly bind to CD80/PD-L1 complexes cd80-cd274-4 1 7 unspecified role unspecified role prey prey purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos Chlorocebus aethiops cos cos 7 competitor competitor competitor prey prey ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos Chlorocebus aethiops cos cos 6 unspecified role unspecified role bait bait c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 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 ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc physical association physical association Fig 3B bottom cd274-cd80-8 1 4 unspecified role unspecified role bait bait ecd sufficient to bind sufficient binding region sufficient to bind range range range range region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 6 unspecified role unspecified role prey prey c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 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 ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc direct interaction direct interaction Supp fig 4-1 cd274-cd80-9 1 7 unspecified role unspecified role bait bait purified purified ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos Chlorocebus aethiops cos cos 4 unspecified role unspecified role prey prey ecd sufficient to bind sufficient binding region sufficient to bind range range range range region fc-igg tag fc-igg tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 6 ancillary ancillary prey prey c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 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 ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc physical association physical association Supp fig 5 not sure if "Biological" Role of Ancillary is OK here - for CD80? CTLA-4 and PD-L1 can bind to human CD80 simultaneously CTLA-4 and PD-L1 can bind to human CD80 simultaneously