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/ li-2016-15 in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Li X., Saha P., Li J., Blobel G., Pfeffer SR. xli05@rockefeller.edu,blobel@rockefeller.edu,pfeffer@stanford.edu Proceedings of the National Academy of Sciences of the United States of America 2016 imex curation Only protein-protein interactions li-2016-16 in vitro In vitro mst microscale thermophoresis mst predetermined predetermined participant predetermined Li X., Saha P., Li J., Blobel G., Pfeffer SR. xli05@rockefeller.edu,blobel@rockefeller.edu,pfeffer@stanford.edu Proceedings of the National Academy of Sciences of the United States of America 2016 imex curation Only protein-protein interactions cholesterol sulfate cholesterol sulfate CHOLEST-5-EN-3-YL HYDROGEN SULFATE Cholest-5-en-3beta-ol sulfate cholesterol sulfate cholesteryl sulfate cholest-5-en-3beta-yl hydrogen sulfate small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) o15118-1 NPC intracellular cholesterol transporter 1 Niemann-Pick C1 protein NPC1 protein protein human Homo sapiens Human MTARGLALGLLLLLLCPAQVFSQSCVWYGECGIAYGDKRYNCEYSGPPKPLPKDGYDLVQELCPGFFFGNVSLCCDVRQLQTLKDNLQLPLQFLSRCPSCFYNLLNLFCELTCSPRQSQFLNVTATEDYVDPVTNQTKTNVKELQYYVGQSFANAMYNACRDVEAPSSNDKALGLLCGKDADACNATNWIEYMFNKDNGQAPFTITPVFSDFPVHGMEPMNNATKGCDESVDEVTAPCSCQDCSIVCGPKPQPPPPPAPWTILGLDAMYVIMWITYMAFLLVFFGAFFAVWCYRKRYFVSEYTPIDSNIAFSVNASDKGEASCCDPVSAAFEGCLRRLFTRWGSFCVRNPGCVIFFSLVFITACSSGLVFVRVTTNPVDLWSAPSSQARLEKEYFDQHFGPFFRTEQLIIRAPLTDKHIYQPYPSGADVPFGPPLDIQILHQVLDLQIAIENITASYDNETVTLQDICLAPLSPYNTNCTILSVLNYFQNSHSVLDHKKGDDFFVYADYHTHFLYCVRAPASLNDTSLLHDPCLGTFGGPVFPWLVLGGYDDQNYNNATALVITFPVNNYYNDTEKLQRAQAWEKEFINFVKNYKNPNLTISFTAERSIEDELNRESDSDVFTVVISYAIMFLYISLALGHMKSCRRLLVDSKVSLGIAGILIVLSSVACSLGVFSYIGLPLTLIVIEVIPFLVLAVGVDNIFILVQAYQRDERLQGETLDQQLGRVLGEVAPSMFLSSFSETVAFFLGALSVMPAVHTFSLFAGLAVFIDFLLQITCFVSLLGLDIKRQEKNRLDIFCCVRGAEDGTSVQASESCLFRFFKNSYSPLLLKDWMRPIVIAIFVGVLSFSIAVLNKVDIGLDQSLSMPDDSYMVDYFKSISQYLHAGPPVYFVLEEGHDYTSSKGQNMVCGGMGCNNDSLVQQIFNAAQLDNYTRIGFAPSSWIDDYFDWVKPQSSCCRVDNITDQFCNASVVDPACVRCRPLTPEGKQRPQGGDFMRFLPMFLSDNPNPKCGKGGHAAYSSAVNILLGHGTRVGATYFMTYHTVLQTSADFIDALKKARLIASNVTETMGINGSAYRVFPYSVFYVFYEQYLTIIDDTIFNLGVSLGAIFLVTMVLLGCELWSAVIMCATIAMVLVNMFGVMWLWGISLNAVSLVNLVMSCGISVEFCSHITRAFTVSMKGSRVERAEEALAHMGSSVFSGITLTKFGGIVVLAFAKSQIFQIFYFRMYLAMVLLGATHGLIFLPVLLSYIGPSVNKAKSCATEERYKGTERERLLNF p61916-1 NPC intracellular cholesterol transporter 2 Human epididymis-specific protein 1 Niemann-Pick disease type C2 protein Epididymal secretory protein E1 NPC2 HE1 protein protein human Homo sapiens Human MRFLAATFLLLALSTAAQAEPVQFKDCGSVDGVIKEVNVSPCPTQPCQLSKGQSYSVNVTFTSNIQSKSSKAVVHGILMGVPVPFPIPEPDGCKSGINCPIQKDKTYSYLNKLPVKSEYPSIKLVVEWQLQDDKNQSLFCWEIPVQIVSHL npc1_mouse NPC intracellular cholesterol transporter 1 Niemann-Pick C1 protein Npc1 protein protein mouse Mus musculus Mouse MGAHHPALGLLLLLLCPAQVFSQSCVWYGECGIATGDKRYNCKYSGPPKPLPKDGYDLVQELCPGLFFDNVSLCCDIQQLQTLKSNLQLPLQFLSRCPSCFYNLMTLFCELTCSPHQSQFLNVTATEDYFDPKTQENKTNVKELEYFVGQSFANAMYNACRDVEAPSSNEKALGLLCGRDARACNATNWIEYMFNKDNGQAPFTIIPVFSDLSILGMEPMRNATKGCNESVDEVTGPCSCQDCSIVCGPKPQPPPPPMPWRIWGLDAMYVIMWVTYVAFLFVFFGALLAVWCHRRRYFVSEYTPIDSNIAFSVNSSDKGEASCCDPLGAAFDDCLRRMFTKWGAFCVRNPTCIIFFSLAFITVCSSGLVFVQVTTNPVELWSAPHSQARLEKEYFDKHFGPFFRTEQLIIQAPNTSVHIYEPYPAGADVPFGPPLNKEILHQVLDLQIAIESITASYNNETVTLQDICVAPLSPYNKNCTIMSVLNYFQNSHAVLDSQVGDDFYIYADYHTHFLYCVRAPASLNDTSLLHGPCLGTFGGPVFPWLVLGGYDDQNYNNATALVITFPVNNYYNDTERLQRAWAWEKEFISFVKNYKNPNLTISFTAERSIEDELNRESNSDVFTVIISYVVMFLYISLALGHIQSCSRLLVDSKISLGIAGILIVLSSVACSLGIFSYMGMPLTLIVIEVIPFLVLAVGVDNIFILVQTYQRDERLQEETLDQQLGRILGEVAPTMFLSSFSETSAFFFGALSSMPAVHTFSLFAGMAVLIDFLLQITCFVSLLGLDIKRQEKNHLDILCCVRGADDGQGSHASESYLFRFFKNYFAPLLLKDWLRPIVVAVFVGVLSFSVAVVNKVDIGLDQSLSMPNDSYVIDYFKSLAQYLHSGPPVYFVLEEGYNYSSRKGQNMVCGGMGCDNDSLVQQIFNAAELDTYTRVGFAPSSWIDDYFDWVSPQSSCCRLYNVTHQFCNASVMDPTCVRCRPLTPEGKQRPQGKEFMKFLPMFLSDNPNPKCGKGGHAAYGSAVNIVGDDTYIGATYFMTYHTILKTSADYTDAMKKARLIASNITETMRSKGSDYRVFPYSVFYVFYEQYLTIIDDTIFNLSVSLGSIFLVTLVVLGCELWSAVIMCITIAMILVNMFGVMWLWGISLNAVSLVNLVMSCGISVEFCSHITRAFTMSTKGSRVSRAEEALAHMGSSVFSGITLTKFGGIVVLAFAKSQIFEIFYFRMYLAMVLLGATHGLIFLPVLLSYIGPSVNKAKRHTTYERYRGTERERLLNF npc2_bovin NPC intracellular cholesterol transporter 2 Epididymal secretory protein E1 NPC2 Niemann Pick type C2 protein homolog EPV20 protein protein bovin Bos taurus (Bovine) Bovine MRFLTVAFLFLALSASALAEPVKFKDCGSWVGVIKEVNVSPCPTQPCKLHRGQSYSVNVTFTSNTQSQSSKAVVHGIVMGIPVPFPIPESDGCKSGIRCPIEKDKTYNYVNKLPVKNEYPSIKVVVEWELTDDKNQRFFCWQIPIEVEA cholesterol sulfate-npc1-2 2876136 N/A 2876138 unspecified role unspecified role unspecified role unspecified role Stoichiometry: 2.0 N/A 2876139 unspecified role unspecified role neutral component neutral component 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 middle_lumenal_domain 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) Stoichiometry: 2.0 N/A 2876140 unspecified role unspecified role neutral component neutral component 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false human-293s Human 293S cell line Human 293s 293S cell line Stoichiometry: 2.0 2876150 2876147 direct interaction direct interaction false false false Supp Table 1 2.4A R work =0.194 R free = 0.249 cholesterol sulfate-npc1-1 2876137 N/A 2876141 unspecified role unspecified role neutral component neutral component preprotrypsin signal sequence tag tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false sequence forming an antiparallel coiled coil tag tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false O35604:p.Phe503_Tyr504delinsAlaAla mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false O35604:p.Asp502_Tyr504delinsAlaAlaAla mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false O35604:p.[Glu421Ala;Phe503_Tyr504delinsAlaAla] mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false n_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range false n_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false middle_lumenal_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false human-293f Homo sapiens HEK293F cell line 293f embryonic kidney cell 293f N/A 2876142 unspecified role unspecified role neutral component neutral component region rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false bovin-milk Bos taurus milk milk N/A 2876138 unspecified role unspecified role neutral component neutral component direct interaction direct interaction false false false 2876137 Fig 3 2.4A Both the D502A,F503A,Y504A and E421F503A,Y504A mutant constructs gave a Kd of >3uM.