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 http://mint.bio.uniroma2.it/mint wang-2014b-2 Structural and mechanistic insights into MICU1 regulation of mitochondrial calcium uptake. in vitro In vitro solution sedimentati cosedimentation in solution solution sedimentati predetermined predetermined participant predetermined Wang L., Yang X., Li S., Wang Z., Liu Y., Feng J., Zhu Y., Shen Y. yuequan74@yahoo.com; yshen@nankai.edu.cn yuequan74@yahoo.com,yshen@nankai.edu.cn EMBO J. (0261-4189) 2014 Only protein-protein interactions imex curation wang-2014b-1 Structural and mechanistic insights into MICU1 regulation of mitochondrial calcium uptake. in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Wang L., Yang X., Li S., Wang Z., Liu Y., Feng J., Zhu Y., Shen Y. yuequan74@yahoo.com; yshen@nankai.edu.cn yuequan74@yahoo.com,yshen@nankai.edu.cn EMBO J. (0261-4189) 2014 Only protein-protein interactions imex curation micu1_human Calcium uptake protein 1, mitochondrial MICU1 Calcium-binding atopy-related autoantigen 1 CBARA1 CALC Atopy-related autoantigen CALC protein protein human Homo sapiens Human MFRLNSLSALAELAVGSRWYHGGSQPIQIRRRLMMVAFLGASAVTASTGLLWKRAHAESPPCVDNLKSDIGDKGKNKDEGDVCNHEKKTADLAPHPEEKKKKRSGFRDRKVMEYENRIRAYSTPDKIFRYFATLKVISEPGEAEVFMTPEDFVRSITPNEKQPEHLGLDQYIIKRFDGKKISQEREKFADEGSIFYTLGECGLISFSDYIFLTTVLSTPQRNFEIAFKMFDLNGDGEVDMEEFEQVQSIIRSQTSMGMRHRDRPTTGNTLKSGLCSALTTYFFGADLKGKLTIKNFLEFQRKLQHDVLKLEFERHDPVDGRITERQFGGMLLAYSGVQSKKLTAMQRQLKKHFKEGKGLTFQEVENFFTFLKNINDVDTALSFYHMAGASLDKVTMQQVARTVAKVELSDHVCDVVFALFDCDGNGELSNKEFVSIMKQRLMRGLEKPKDMGFTRLMQAMWKCAQETAWDFALPKQ micu1-1 1547348 n/a 1547350 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 6.0 direct interaction direct interaction false false false sf3a f2a In the absence of Ca2+, MICU1-xtal in solution predominantly existed as a hexamer (~237 kDa, theoretical molecular weight of 43 kDa for each molecule) (Fig 2A and Supplementary Fig 3A), whereas, in the presence of Ca2+, MICU1-xtal existed as a mixture of oligomeric forms (Fig 2B and Supplementary Fig 3B). micu1-micu1 1547348 n/a 1547350 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false n/a 1547350 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false direct interaction direct interaction false false false sf3b f2b In the absence of Ca2+, MICU1-xtal in solution predominantly existed as a hexamer (~237 kDa, theoretical molecular weight of 43 kDa for each molecule) (Fig 2A and Supplementary Fig 3A), whereas, in the presence of Ca2+, MICU1-xtal existed as a mixture of oligomeric forms (Fig 2B and Supplementary Fig 3B). micu1-1 1547348 n/a 1547350 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Q9BPX6:p.Arg221Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 direct interaction direct interaction false false false sf3c f2e f2f f3f f3g To verify this hypothesis, we determined the oligomerization state of MICU1-xtal-deltaC. This mutant exhibited a predominant peak corresponding to the MICU1 dimer both in the absence and in the presence of Ca2+ (Fig 2E, F and Supplementary Fig 3C), suggesting that the C-helix is necessary for the formation of the Ca2+-free MICU1-xtal hexamer and substantially contributed to the formation of multiple oligomers of Ca2+-bound MICU1-xtal. Within the Ca2+-free MICU1-xtal dimer interface, the two monomers primarily interact through a salt bridge. Asp376 from one monomer interacts with Arg221 from the reciprocal monomer via a salt bridge (Fig 3D). The R221A or D376A single mutation or the R221AD376A double mutation of MICU1-xtal-deltaC converted the dimer into the monomer in the absence of Ca2+ (Fig 3F). The Ca2+-bound MICU1-xtal-deltaC dimer interface is formed by hydrogen bonding and hydrophobic interactions (Fig 3E) The R221A single mutation of MICU1- xtal-deltaC was unable to disrupt the dimer formation in the presence of Ca2+. The F383A-H385A double mutation of MICU1-xtal-deltaC converted the dimer into the monomer, and the dimer mixture had an approximate ratio of 1:1 in the presence of Ca2+ (Fig 3G). micu1-1 1547348 n/a 1547350 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false Q9BPX6:p.[Arg221Ala;Asp376Ala] mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 direct interaction direct interaction false false false sf3c f2e f2f f3f f3g To verify this hypothesis, we determined the oligomerization state of MICU1-xtal-deltaC. This mutant exhibited a predominant peak corresponding to the MICU1 dimer both in the absence and in the presence of Ca2+ (Fig 2E, F and Supplementary Fig 3C), suggesting that the C-helix is necessary for the formation of the Ca2+-free MICU1-xtal hexamer and substantially contributed to the formation of multiple oligomers of Ca2+-bound MICU1-xtal. Within the Ca2+-free MICU1-xtal dimer interface, the two monomers primarily interact through a salt bridge. Asp376 from one monomer interacts with Arg221 from the reciprocal monomer via a salt bridge (Fig 3D). The R221A or D376A single mutation or the R221AD376A double mutation of MICU1-xtal-deltaC converted the dimer into the monomer in the absence of Ca2+ (Fig 3F). The Ca2+-bound MICU1-xtal-deltaC dimer interface is formed by hydrogen bonding and hydrophobic interactions (Fig 3E) The R221A single mutation of MICU1- xtal-deltaC was unable to disrupt the dimer formation in the presence of Ca2+. The F383A-H385A double mutation of MICU1-xtal-deltaC converted the dimer into the monomer, and the dimer mixture had an approximate ratio of 1:1 in the presence of Ca2+ (Fig 3G). micu1-1 1547348 n/a 1547350 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Q9BPX6:p.Asp376Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 direct interaction direct interaction false false false sf3c f2e f2f f3f f3g To verify this hypothesis, we determined the oligomerization state of MICU1-xtal-deltaC. This mutant exhibited a predominant peak corresponding to the MICU1 dimer both in the absence and in the presence of Ca2+ (Fig 2E, F and Supplementary Fig 3C), suggesting that the C-helix is necessary for the formation of the Ca2+-free MICU1-xtal hexamer and substantially contributed to the formation of multiple oligomers of Ca2+-bound MICU1-xtal. Within the Ca2+-free MICU1-xtal dimer interface, the two monomers primarily interact through a salt bridge. Asp376 from one monomer interacts with Arg221 from the reciprocal monomer via a salt bridge (Fig 3D). The R221A or D376A single mutation or the R221AD376A double mutation of MICU1-xtal-deltaC converted the dimer into the monomer in the absence of Ca2+ (Fig 3F). The Ca2+-bound MICU1-xtal-deltaC dimer interface is formed by hydrogen bonding and hydrophobic interactions (Fig 3E) The R221A single mutation of MICU1- xtal-deltaC was unable to disrupt the dimer formation in the presence of Ca2+. The F383A-H385A double mutation of MICU1-xtal-deltaC converted the dimer into the monomer, and the dimer mixture had an approximate ratio of 1:1 in the presence of Ca2+ (Fig 3G). micu1-1 1547348 n/a 1547350 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false Q9BPX6:p.[Phe383Ala;His385Ala] mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 direct interaction direct interaction false false false sf3c f2e f2f f3f f3g To verify this hypothesis, we determined the oligomerization state of MICU1-xtal-deltaC. This mutant exhibited a predominant peak corresponding to the MICU1 dimer both in the absence and in the presence of Ca2+ (Fig 2E, F and Supplementary Fig 3C), suggesting that the C-helix is necessary for the formation of the Ca2+-free MICU1-xtal hexamer and substantially contributed to the formation of multiple oligomers of Ca2+-bound MICU1-xtal. Within the Ca2+-free MICU1-xtal dimer interface, the two monomers primarily interact through a salt bridge. Asp376 from one monomer interacts with Arg221 from the reciprocal monomer via a salt bridge (Fig 3D). The R221A or D376A single mutation or the R221AD376A double mutation of MICU1-xtal-deltaC converted the dimer into the monomer in the absence of Ca2+ (Fig 3F). The Ca2+-bound MICU1-xtal-deltaC dimer interface is formed by hydrogen bonding and hydrophobic interactions (Fig 3E) The R221A single mutation of MICU1- xtal-deltaC was unable to disrupt the dimer formation in the presence of Ca2+. The F383A-H385A double mutation of MICU1-xtal-deltaC converted the dimer into the monomer, and the dimer mixture had an approximate ratio of 1:1 in the presence of Ca2+ (Fig 3G). micu1-1 1547349 n/a 1547350 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 6.0 direct interaction direct interaction false false false f1 f2c t1 sf4 Consistent with the observations in solution, the Ca2+-free MICU1-xtal crystal structure revealed six molecules in the asymmetric unit, which are packed by a trimer of dimers (Fig 2C and Supplementary Fig 4). Unexpectedly, in the Ca2+-free MICU1-xtal structure, the C-helix is located in the center of the six molecules that are packed as a helix bundle. micu1-1 1547349 n/a 1547350 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified range binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 direct interaction direct interaction false false false f3 t1 Ca2+ binding to MICU1 induced large conformational changes in the monomer, which led to a completely different dimer formation. The interface of the Ca2+-bound MICU1-xtal-deltaC dimer was composed of the CH4 helix from both monomers as well as the CH4-CH5 loop from one monomer with the NH3 helix from the reciprocal monomer. The buried surface area of the Ca2+-bound MICU1-xtal-deltaC dimer was approximately 434 A2 (Fig 3B). The overall dimers of the Ca2+-free and the Ca2+-bound MICU1 structures could not be superposed. After the superposition of each monomer from the two dimers, the additional superposition of the second monomer from the two dimers required an approximately 74° rotation (Fig 3C).