mpidb The Microbial Protein Interaction Database fu-2007-1 Characterization of MazFSa, an endoribonuclease from Staphylococcus aureus. ecolx Escherichia coli pull down pull down weight by comassie molecular weight estimation by coomasie staining weight by comassie Fu Z., Donegan N.P., Memmi G., Cheung A.L. J. Bacteriol. (0021-9193) 2007 Only protein-protein interactions imex curation fu-2007-2 Characterization of MazFSa, an endoribonuclease from Staphylococcus aureus. in vitro In vitro bn-page blue native page bn-page weight by comassie molecular weight estimation by coomasie staining weight by comassie Fu Z., Donegan N.P., Memmi G., Cheung A.L. J. Bacteriol. (0021-9193) 2007 Only protein-protein interactions imex curation maze_staae Antitoxin MazE mazE NWMN_1974.1 protein protein staae Staphylococcus aureus (strain Newman) MLSFSQNRSHSLEQSLKEGYSQMADLNLSLANEAFPIECEACDCNETYLSSNSTNE mazf_staae Endoribonuclease MazF MazFSa mRNA interferase MazF NWMN_1974 mazF Toxin MazF protein protein staae Staphylococcus aureus (strain Newman) MIRRGDVYLADLSPVQGSEQGGVRPVVIIQNDTGNKYSPTVIVAAITGRINKAKIPTHVEIEKKKYKLDKDSVILLEQIRTLDKKRLKEKLTYLSDDKMKEVDNALMISLGLNAVAHQKN mazf-maze 1332181 n/a 1332183 unspecified role unspecified role prey prey n/a 1332184 unspecified role unspecified role bait bait n-terminal 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 physical association physical association false false false Fig. 1D maze-mazf-1 1332182 n/a 1332183 unspecified role unspecified role neutral component neutral component n-terminal 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 ecolx Escherichia coli n/a 1332184 unspecified role unspecified role neutral component neutral component n-terminal 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 ecolx Escherichia coli physical association physical association false false false Fig. 1E 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/ casino-2009-3 Structural insight into partner specificity and phosphoryl transfer in two-component signal transduction. in vitro In vitro protein kinase assay protein kinase assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Casino P., Rubio V., Marina A. Phosphorylation monitored by incorporation of 32P-labelled phosphate onto protein. amarina@ibv.csic.es Cell (0092-8674) 2009 Only protein-protein interactions 9 imex curation q9wzv7_thema TM_0853 protein protein thema Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099) MEEIKEQNNRLFLLLSIMIMEFSNAKTESDILQGLLNLVRKVVDIKEVVLVDENKRKIWGRDIDIKRFEEFIDWSIRQSNPVFVEDELGYVGIVPVVKQDRMFGSLIVLLNHQPSMEETEIFKVLSFLSAVVLENIKLYRELEETYNYVNVILNGLPEGIFVYSNGEIKFQNEKFKEENFPDEVLRKAISLSEEAISLRTQRVGEVISGEEFFSITSIPLILGSEVQALTIVENVTESKELERLKRIDRMKTEFIANISHELRTPLTAIKAYAETIYNSLGELDLSTLKEFLEVIIDQSNHLENLLNELLDFSRLERKSLQINREKVDLCDLVESAVNAIKEFASSHNVNVLFESNVPCPVEAYIDPTRIRQVLLNLLNNGVKYSKKDAPDKYVKVILDEKDGGVLIIVEDNGIGIPDHAKDRIFEQFYRVDSSLTYEVPGTGLGLAITKEIVELHGGRIWVESEVGKGSRFFVWIPKDRAGEDNRQDN phor_bacsu Alkaline phosphatase synthesis sensor protein PhoR phoR BSU29100 protein protein bacsu Bacillus subtilis MNKYRVRLFSVFVVCMILVFCVLGLFLQQLFETSDQRKAEEHIEKEAKYLASLLDAGNLNNQANEKIIKDAGGALDVSASVIDTDGKVLYGSNGRSADSQKVQALVSGHEGILSTTDNKLYYGLSLRSEGEKTGYVLLSASEKSDGLKGELWGMLTASLCTAFIVIVYFYSSMTSRYKRSIESATNVATELSKGNYDARTYGGYIRRSDKLGHAMNSLAIDLMEMTRTQEMQRDRLLTVIENIGSGLIMIDGRGFINLVNRSYAKQFHINPNHMLRRLYHDAFEHEEVIQLVEDIFMTETKKCKLLRLPIKIERRYFEVDGVPIMGPDDEWKGIVLVFHDMTETKKLEQMRKDFVANVSHELKTPITSIKGFTETLLDGAMEDKEALSEFLSIILKESERLQSLVQDLLDLSKIEQQNFTLSIETFEPAKMLGEIETLLKHKADEKGISLHLNVPKDPQYVSGDPYRLKQVFLNLVNNALTYTPEGGSVAINVKPREKDIQIEVADSGIGIQKEEIPRIFERFYRVDKDRSRNSGGTGLGLAIVKHLIEAHEGKIDVTSELGRGTVFTVTLKRAAEKSA a0a0h3k900_staae Sensor protein kinase WalK phoR NWMN_1585 protein protein staae Staphylococcus aureus (strain Newman) MMKFHHRLMLLISSILIISFVTLGIIIHASITYVLSEKHEQALATDARSYIYLVQDNEIDKVKKIVKQQNIDLLITKHDKKVFSSGKLKAIPNSDSYIQKRDVFIFNKKVNGYHVWIKGYNNDITEMHWTLWKYLILTCLVVLICLYFASRSFKRTLIRPIQEVTYATQLLANGYYHIRVPESNVVETKALFVSTNDLARRLQKLNNEQKIQSNRLKTTIENIPSAILMIDRNGKIVVANKAYYEQFNISHNIEQVGYHGYVNTEIEQLILESFKVEKPIYEQLEVAINQVHAKYFDISCVPILTRSQKSLQGILVVMHDITNLKQLENLRREFVANVSHELKTPITSIKGFAETLIDGAKNDAESLDMFLNIILKESNRIESLVTDLLDLSHIEQHTELDTDYMNLSDLTRRIIDNMMTQANQKNISIHTDIEKDVIVKAQESKIAQVITNLLTNAINYSYEDGDINVRVYRDDFRVIFEVQDFGIGIKLEDQQRIFERFYRVDKARSRDSGGTGLGLSITKHIVEAHQGNIEVNSQVGKGSTFKVILKDYKE q9wzv7-1 Autophosphorylation of Q9WZV7 (HK853) 860113 n/a 860117 self self self self region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c-terminal 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 ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 phosphorylation phosphorylation reaction phosphorylation false true false Fig. 3C, 5A-E q9wzv7-2 Autophosphorylation of Q9WZV7 (HK853) 860113 n/a 860117 putative self putative self putative self putative self Q9WZV7:p.His260Ala Alters kinetics of interaction mutation mutation certain certain sequence position certain certain certain sequence position certain false c-terminal 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 n/a 860117 putative self putative self putative self putative self region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c-terminal 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 Q9WZV7:p.His260Ala Alters kinetics of interaction mutation mutation certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation false false false Fig. 3C phor-1 Autophosphorylation of PhoR 860113 n/a 860118 self self self self cytoplasmic 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) phosphorylation phosphorylation reaction phosphorylation false true false Fig. 5F phor-2 Autophosphorylation of PhoR 860113 n/a 860119 putative self putative self putative self putative self ecoli Escherichia coli (strain K12) n/a 860119 putative self putative self putative self putative self region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n-terminal 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 ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation false false false Fig. 5F phor-3 Autophosphorylation of PhoR 860113 n/a 860118 putative self putative self putative self putative self cytoplasmic 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 860118 putative self putative self putative self putative self cytoplasmic region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n-terminal 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 A0A0H3K900:p.Asn456Ala Alters kinetics of interaction mutation mutation certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation false false false Fig. 5F 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 jger-2016-1 in vitro In vitro crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Jäger F., Zoltner M., Kneuper H., Hunter WN., Palmer T. tracy.palmer@dundee.ac.uk FEBS letters 2016 Only protein-protein interactions imex curation imex curation jger-2016-2 in vitro In vitro bn-page blue native page bn-page western blot western blot Immuno blot Immuno blot Jäger F., Zoltner M., Kneuper H., Hunter WN., Palmer T. tracy.palmer@dundee.ac.uk FEBS letters 2016 Only protein-protein interactions imex curation imex curation essc_staae Type VII secretion system protein EssC essC NWMN_0223 protein protein staae Staphylococcus aureus (strain Newman) MHKLIIKYNKQLKMLNLRDGKTYTISEDERADITLKSLGEVIHLEQNNQGTWQANHTSINKVLVRKGDLDDITLQLYTEADYASFAYPSIQDTMTIGPNAYDDMVIQSLMNAIIIKDFQSIQESQYVRIVHDKNTDVYINYELQEQLTNKAYIGDHIYVEGIWLEVQADGLNVLSQNTVASSLIRLTQEMPHAQADDYNTYHRSPRIIHREPTDDIKIERPPQPIQKNNTVIWRSIIPPLVMIALTVVIFLVRPIGIYILMMIGMSTVTIVFGITTYFSEKKKYNKDVEKREKDYKAYLDNKSKEINKAIKAQRFSLNYHYPTVAEIKDIVETKAPRIYEKTSHHHDFLHYKLGIANVEKSFKLDYQEEEFNQRRDELFDDAKELYEFYTDVEQAPLINDLNHGPIAYIGARHLILEELEKMLIQLSTFHSYHDLEFLFVTREDEVETLKWARWLPHMTLRGQNIRGFVYNQRTRDQILTSIYSMIKERIQAVRERSRSNEQIIFTPQLVFVITDMSLIIDHVILEYVNQDLSEYGISLIFVEDVIESLPEHVDTIIDIKSRTEGELITKEKELVQLKFTPENIDNVDKEYIARRLANLIHVEHLKNAIPDSITFLEMYNVKEVDQLDVVNRWRQNETYKTMAVPLGVRGKDDILSLNLHEKAHGPHGLVAGTTGSGKSEIIQSYILSLAINFHPHEVAFLLIDYKGGGMANLFKDLVHLVGTITNLDGDEAMRALTSIKAELRKRQRLFGEHDVNHINQYHKLFKEGIATEPMPHLFIISDEFAELKSEQPDFMKELVSTARIGRSLGIHLILATQKPSGVVDDQIWSNSKFKLALKVQDRQDSNEILKTPDAADITLPGRAYLQVGNNEIYELFQSAWSGATYDIEGDKLEVEDKTIYMINDYGQLQAINKDLSGLEDEETKENQTELEAVIDHIESITTRLEIEEVKRPWLPPLPENVYQEDLVETDFRKLWSDDAKEVELTLGLKDVPEEQYQGPMVLQLKKAGHIALIGSPGYGRTTFLHNIIFDVARHHRPDQAHMYLFDFGTNGLMPVTDIPHVADYFTVDQEDKIAKAIRIFNDEIDRRKKILSQYRVTSISEYRKLTGETIPHVFILIDNFDAVKDSPFQEVFENMMIKMTREGLALDMQVTLTASRANAMKTPMYINMKTRIAMFLYDKSEVSNVVGQQKFAVKDVVGRALLSSDDNVSFHIGQPFKHDETKSYNDQINDEVSAMTEFYKGETPNDIPMMPDEIKYEDYRESLNLPDIVANGALPIGLDYEGVTLQKIKLTEPAMISSENPREIAHIAEIMMKEIDILNEKYAICIADSSGEFKAYRHQVANFAEEREDIKAIHQLMIEDLKQREMDGPFEKDSLYIINDFKTFIDCTYIPEDDVKKLITKGPELGLNILFVGIHKELIDAYDKQIDVARKMINQFSIGIRISDQQFFKFRFIQREPVIKENEAYMVANQAYQKIRWFK esaa_staae Type VII secretion system accessory factor EsaA esaA NWMN_0220 protein protein staae Staphylococcus aureus (strain Newman) MKKKNWIYALIVTLIIIIAIVSMIFFVQTKYGDQSEKGSQSVSNKNNKIHIAIVNEDQPTTYNGKKVELGQAFIKRLANEKNYKFETVTRNVAESGLKNGGYQVMIVIPENFSKLAMQLDAKTPSKISLQYKTAVGQKEEVAKNTEKVVSNVLNDFNKNLVEIYLTSIIDNLHNAQKNVGAIMTREHGVNSKFSNYLLNPINDFPELFTDTLVNSISANKDITKWFQTYNKSLLSANSDTFRVNTDYNVSTLIEKQNSLFDEHNTAMDKMLQDYKSQKDSVELDNYINALKQMDSQIDQQSSMQDTGKEEYKQTVKENLDKLREIIQSQESPFSKGMIEDYRKQLTESLQDELANNKDLQDALNSIKMNNAQFAENLEKQLHDDIVKEPDTDTTFIYNMSKQDFIAAGLNEGEANKYEAIVKEAKRYKNEYNLKKPLAEHINLTDYDNQVAQDTSSLINDGVKVQRTETIKSNDINQLTVATDPHFNFEGDIKINGKKYDIKDQSVQLDTSNKEYKVEVNGVAKLKKDAEKDFLKDKTMHLQLLFGQANRQDEPNDKKATSVVDVTLNHNLDGRLSKDALSQQLSALSRFDAHYKMYTDTKGREDKPFDNKRLIDMMVDQVINDMESFKDDKVAVLHQIDSMEENSDKLIDDILNNKKNTTKNKEDISKLIDQLENVKKTFAEEPQEPKIDKGKNDEFNTMSSNLDKEISRISEKSTQLLSDTQESKSIADSVSGQLNQVDNNVNKLHATGRALGVRANDLNRQMAKNDKDNELFAKEFKKVLQNSKDGDRQNQALKAFMSNPVQKKNLENVLANNGNTDVISPTLFVLLMYLLSMITAYIFYSYERAKGQMNFIKDDYSSKNHLWNNVITSGVIGTTGLVEGLIVGLIAMNKFHVLAGYRAKFILMVILTMMVFVLINTYLLRQVKSIGMFLMIAALGLYFVAMNNLKAAGQGVTNKISPLSYIDNMFFNYLNAEHPIGLVLVILTVLVIIGFVLNMFIKHFKKERLI essb_staae Type VII secretion system protein EssB NWMN_0222 essB protein protein staae Staphylococcus aureus (strain Newman) MVKNHNPKNEMQDMLTPLDAEEAAKTKLRLDMREIPKSSIKPEHFHLMYLLEQHSPYFIDAELTELRDSFQIHYDINDNHTPFDNIKSFTKNEKLRYLLNIKNLEEVNRTRYTFVLAPDELFFTRDGLPIAKTRGLQNVVDPLPVSEAEFLTRYKALVICAFNEKQSFDALVEGNLELHKGTPFETKVIEAATLDLLTAFLDEQYQKQEQDYSQNYAYVRKVGHTVFKWVAIGMTTLSVLLIAFLAFLYFSVMKHNERIEKGYQAFVKDDYTQVLNTYDDLDGKKLDKEALYIYAKSYIQTNKQGLEKDKKENLLNNVTPNSNKDYLLYWMELGQGHLDEAINIATYLDDNDITKLALINKLNEIKNNGDLSNDKRSEETKKYNDKLQDILDKEKQVKDEKAKSEEEKAKAKDEKLKQQEENEKKQKEQAQKDKEKRQEAERKK essa-1 1929557 n/a 1929559 unspecified role unspecified role neutral component neutral component subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level In vivo crosslinking analysis of EssB resulted in no detectable EssB crosslinks unless the protein was overproduced, in which case a very faint crosslinked product running at the approximate size of an EssB homo-dimer could be detected (Fig 3B). As homo-dimerisation of EssB has been reported previously, [15, 16] it is likely that this crosslink is a homo-dimeric form of EssB. direct interaction direct interaction false false false f2 f3 essa-2 1929557 n/a 1929560 unspecified role unspecified role neutral component neutral component subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level direct interaction direct interaction false false false f3 As shown in Fig. 3A, incubation with formaldehyde resulted in EsaA-specific crosslinks in whole cells of the wild-type strain. A series of 4–5 discrete bands could be detected, migrating around 250 kDa. Interestingly, essentially the same pattern of EsaA crosslinks was seen whether EssA, EssB or EssC were absent, suggesting that the crosslinked products did not contain any of these proteins. It has been reported that the long extracellular domain of YueB, the Bacillus subtilis homologue of EsaA, forms a highly elongated homo-dimer [17] and it is therefore likely that the crosslinks seen here correspond to different conformers of a crosslinked EsaA dimer. essc-1 1929557 n/a 1929561 unspecified role unspecified role neutral component neutral component subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level N/A 1929561 unspecified role unspecified role neutral component neutral component subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level direct interaction direct interaction false false false f3 Crosslinking of EssC in vivo also yielded high molecular weight products, as was observed in vitro (Fig 3C). However, in contrast to the in vitro analysis, where one major crosslinked product was seen, in this instance at least two distinct high molecular weight species were detected, both of which migrated above the 460 kDa molecular weight marker. The differences between the pattern of crosslinks seen in vivo and in vitro may reflect the difference in size between DSS which was used as a crosslinker in vitro and formaldehyde which was used in vivo. Alternatively they may arise due to the presence of ATP, proton-motive force or other factors that are present in whole cells. It should be noted that as similar crosslinks were seen in the absence of any of EsaA, EssA or EssB, neither of the two crosslinks arise from interactions of EssC with any of the other core membrane components of the Ess system, and probably represent homo-multimeric forms of EssC. essa-3 1929558 n/a 1929559 unspecified role unspecified role neutral component neutral component subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level direct interaction direct interaction false false false f5 However, bands containing EsaA and EssB were observed. As shown in Fig. 5(A), two complexes containing EsaA could be detected, one of which close to the 150 kDa marker and the second just above the 200 kDa marker, which could conceivably correspond to monomers and dimers of EsaA. In contrast, a single EssB-containing band was detected, migrating between the 66 kDa and 150 kDa marker, close to the expected size for an EssB dimer (Fig. 5B). Thus, given the differences in the masses of these bands these two proteins are highly unlikely to be part of the same complex. We confirmed this by repeating the BN PAGE on Fos-choline-12 membrane extracts from strains deleted for esaA or essB, and detected the same two EsaA complexes in the absence of EssB, and the same EssB complex in the absence of EsaA. We conclude that these two proteins do not interact with each other in isolated membranes. esaa-1 1929558 n/a 1929560 unspecified role unspecified role neutral component neutral component subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level direct interaction direct interaction false false false f5 However, bands containing EsaA and EssB were observed. As shown in Fig. 5(A), two complexes containing EsaA could be detected, one of which close to the 150 kDa marker and the second just above the 200 kDa marker, which could conceivably correspond to monomers and dimers of EsaA. In contrast, a single EssB-containing band was detected, migrating between the 66 kDa and 150 kDa marker, close to the expected size for an EssB dimer (Fig. 5B). Thus, given the differences in the masses of these bands these two proteins are highly unlikely to be part of the same complex. We confirmed this by repeating the BN PAGE on Fos-choline-12 membrane extracts from strains deleted for esaA or essB, and detected the same two EsaA complexes in the absence of EssB, and the same EssB complex in the absence of EsaA. We conclude that these two proteins do not interact with each other in isolated membranes.