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 Structure-function analysis of the ATP-driven glycolipid efflux pump DevBCA reveals complex organization with TolC/HgdD. Structure-function analysis of the ATP-driven glycolipid efflux pump DevBCA reveals complex organization with TolC/HgdD. FEBS Lett. (0014-5793) 2013 imex curation Staron P., Forchhammer K., Maldener I. iris.maldener@uni-tuebingen.de 18-Feb-2014: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined iris.maldener@uni-tuebingen.de Accepted 2013-DEC-04 by LUANA 2013 imex curation Structure-function analysis of the ATP-driven glycolipid efflux pump DevBCA reveals complex organization with TolC/HgdD. Structure-function analysis of the ATP-driven glycolipid efflux pump DevBCA reveals complex organization with TolC/HgdD. FEBS Lett. (0014-5793) 2013 imex curation Staron P., Forchhammer K., Maldener I. iris.maldener@uni-tuebingen.de 18-Feb-2014: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined iris.maldener@uni-tuebingen.de Accepted 2013-DEC-04 by LUANA 2013 imex curation q8yt39_noss1 alr2887 protein protein noss1 Nostoc sp. (strain PCC 7120 / UTEX 2576) MKGQHLFYSFLPGVTAAVLTTQPAVAEAMKVSSVQLVPSSSVVATQNSQTLLVDKSQTQLPNATVQIFPDIIPTSGVTTPNLQFSSSHSHPIILTENTGIDREQKSRQDEVKNFPLKSKLTLSQRLKQRRFSSQQKQINSGITKPKLVTKVVSTTSSTLLAGQQTFSPSYLQNPAPQEKLKTQLPMLLQTKANHVGAAKLLNISSCSPQTCQLTKLTGQQIAQGNTTQDPASVPVPDESTLVDTQAPTDTPVPNESAPTDTPVSTESAPADTQSPRVAPVQTESAPVNTQAPAGAVEVPQNLIPNANPLQFPTKPEEVTLKENQPITLTQALELARRSNRDLQVSLLELQRTQALLKEAQAALLPNLSVSSDVTRSQSASGQLQDELISKQGGVGNNDEPGTAFNSQAQLSYDLYTSGRRKAAIQEAEEQVRFNELDVERQSEEIRLSVTRAYYDLQQADEQVRIARSAVENAQASLRDAEALERAGVGTRFDVLRSQVNLANNTQQLTNNLSRQQTTRRQLATLLSVPQAVNISAADPVQLAGLWNQSLEQSIILAYQNRPELHQQLAQRNISEQQRRQALATLGPQVSFVANYNLLDQFDDGVGLTDGYSVGVRATLNLYDGGAARARASQARANIAIAETRFASQRDQVRFQVEQAFTNQRSNLENVQTANVALEQAREALRLARLRFQAGVGTQTDVINSENDLTQAEGNRITAILDYNRALAELQRAVTSRALISSQ 1A25E6AEE4E1010F q7a2a8_noss1 devA protein protein noss1 Nostoc sp. (strain PCC 7120 / UTEX 2576) MRQEAVIAIKSLNHYYGKGALKRQILFDINLEIYPGEIVIMTGPSGSGKTTLLSLIGGLRSVQEGNLQFLGVELSGASQNKLVQIRRSIGYIFQAHNLLGFLTARQNVQMAVELNEHISQEEAIAKAEAMLTAVGLENRVDYYPENLSGGQKQRVAIARALVNNPPLVLADEPTAALDKQSGRDVVEIMQRLAKDQGTSILLVTHDNRILDIADRIVEMEDGILARDSQTAIVSYDSGAWNETP 34182185A65C0A4B q7a2a9_noss1 devC protein protein noss1 Nostoc sp. (strain PCC 7120 / UTEX 2576) MNRKRIPLSWLQLTREKTRLAVALAGIAFADILMFMQIGFRDALYYSNVRLHTSLQGDIVLLNKQSNAVLAMKPFSQRRLYKSLELPSVQSVHPIYLDYSIWKNPYTGLTRSILVFGINPETNIFNLAGVQENLDKLKLPDTVLFDRSSRPEYGPIAKNFEQGQSISAEVRRRRINVVGLFTLGASFGADGNLVTSDVNFLRLFPLRRQGLIDIGLIRLKPGVNPNTAVQELRSYLPDDINVLTKQEFIDFERNYWASSTAIGFIFTLGTIMGFIVGTVIVYQILYTEVADHLSEYATLKAIGYTQKYLLGVILQEALLLACLGYLPGWIFAIILYKTARQATLLPVFMSYDRAITVLILTMIMCFVSGSIAVRKLRSADPADIF 8038F00980FE288E q7a2b0_noss1 devB protein protein noss1 Nostoc sp. (strain PCC 7120 / UTEX 2576) MSRVTEERRSREQPLDLEQPKIWWGLAVALPVAVAAGLLATAKFEQLKRLGPSVPVKPVITSVSAVGRLEPQGEVIKLSAPVGGMQSASRVKQLFVKEGERVRKGQVIAILDNHDTQLAAVEEAKAKLLESRANLAQVRVGSPRDIQAQTAVIARLQAQLVGEMGAQQASITRIASQLSGEKVAQQALVNRLEAELVGQQDSLRATLNRIRAEQRNAQVDAGRYDFLYREGAISQQERDRRRLTATTANQQVIESQAALRQALATLRQQVAEARANQMKTLASLQQQLIEARVTRDKTITTLQRQIDEEKARLKRLVEVSPTDVQMAQAQVSNAIANVRRAEAELKLSYIQAPSAGEILKIYSKSGEAITADGIAEMGETEQMMIIAEVPEDSIGRVRIGQTVTANSDNGAFSGEIRGTVTEIGRKIGKKDVLNTDPAADVDARVVEVKIALSPEDSQKVSGLTYAKVVVDINK 8A1802685ABE7F2D devb 2 DevB 6 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain direct interaction direct interaction f2a sf3a In SEC, DevB appears to be in an oligomeric equilibrium between monomers, dimers, trimers and hexamers. The majority of reference DevB (Fig. 1D and Tab. 1) was eluted as hexamer or monomer (Fig. 2A; upper solid line). In contrast, hexamer formation of DevB lacking the entire α-helical domain (B-αHD in Fig. 1D and Tab. 1) was hardly observable (Fig. 2A; lower solid line). This DevB variant could not efficiently interact with immobilized reference DevB in SPR (Fig. 2B; B-αHD). Removing the distal part of DevB´s α-helical domain (B-DαHD in Fig. 1D and Tab. 1) showed no remarkable effect on hexamer formation as observed in SEC (Fig. 2A; dashed line), and B-DαHD retained ~80% of interaction with immobilized reference DevB, as compared to reference DevB itself as prey (Fig. 2B; B and B-DαHD). However, deleting the proximal part of DevB´s α-helical domain (B-PαHD; Fig. 1D and Tab. 1) decreased this interaction to ~25% (Fig. 2B; B-PαHD). This variant did not form substantial amounts of hexamers (Fig. 2A; dotted line). DevB lacking the β-barrel domain (B-βBD; Fig. 1D and Tab. 1) formed ~75% less hexamers than reference DevB (Fig. 2A; dot-dot-dashed line). Replacing central parts of the lipoyl domain (BxLipD in Fig. 1D and Tab. 1) led to hardly detectable DevB hexamers (Fig. 2A; dot-dashed line). Consistent with these data, both constructs had decreased affinities to immobilized reference DevB (Fig. 2B; B-βBD at ~45% and BxLipD at ~30%). In summary, all domains of DevB, except the distal part of the α-helical domain, are required for stabilization of the inter-MFP interactions, and therefore facilitate hexamer formation. devb-devb 1 DevB 6 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role prey prey purified purified binding region binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain DevB 6 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role bait bait purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f2b devb-q8yt39 1 TolC 3 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role bait bait purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined DevB 6 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role prey prey purified purified binding region binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain direct interaction direct interaction f3a sf5a all DevB’s lacking the native α-helical tips (B*, B-DαHD*, 137 B-PαHD*, BxLipD*, and B-βBD*; see Fig. 1D and Tab. 1) could hardly interact with immobilized 138 reference TolC in SPR experiments (Fig. 3A). The affinities of native α-helical tip variants of DevB 139 toward TolC (B-DαHD > B-βBD > B-PαHD, BxLipD > B-αHD; Fig. 3A) correlated well with the 140 ability to hexamerize (B-DαHD > B-βBD > B-PαHD, BxLipD > B-αHD; Fig. 2). devb-q8yt39-1 1 TolC 3 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role prey prey purified purified Q8YT39:p.Arg688_Val695delinsSerSerGlySerSerGlySerSer mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain RLRFQAGV SSGSSGSS Fri Jul 22 11:14:39 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'agr688ser,leu689ser,arg690gly,phe691ser,gln692ser,ala693gly,gly694ser,val695ser' Wed May 02 18:22:07 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg_leu_arg_phe_gln_ala_gly_val688-695ser_ser_gly_ser_ser_gly_ser_ser' Tue Jun 30 20:23:28 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg688_Val695delinsSerSerGlySerSerGlySerSer' Q8YT39:p.Arg478_Arg485delinsGlyGlySerGlyGlySerGlyGly mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain RDAEALER GGSGGSGG Fri Jul 22 10:59:42 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'arg478gly,asp479gly,ala480ser,glu481gly,ala482gly,leu483ser,glu484gly,arg485gly' Wed May 02 19:08:30 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg_asp_ala_glu_ala_leu_glu_arg478-485gly_gly_ser_gly_gly_ser_gly_gly' Tue Jun 30 19:51:29 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg478_Arg485delinsGlyGlySerGlyGlySerGlyGly' DevB 6 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role bait bait purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f3b sf5b sf2 We also investigated structural criteria of TolC required for binding to DevB. We quantified the interaction of TolC variants lacking the native α-helical tips (replaced by GGS/SSG; Fig. 1C) with immobilized reference DevB via SPR analysis. In line with the effect of tip-mutations of DevB (Fig. 3A, constructs with *), tip-mutated TolC could not efficiently interact with surface-bound reference DevB in SPR experiments (Fig. 3B; D*145 ). deva-devb 1 DevA 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role unspecified role unspecified role purified purified DevB 6 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role prey prey purified purified binding region binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain DevC 5 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role unspecified role unspecified role purified purified physical association physical association f4a sf6 The DevB variants lacking the proximal or the entire α-helical domain (B-αHD and B-PαHD; Fig. 1D and Tab. 1) were impaired in interacting with immobilized DevAC by ~70% (Fig. 4A). Mutating 168 the lipoyl domain or the β-barrel domain (BxLipD and B-βBD; Fig. 1D and Tab. 1) resulted in a decrease of 90% as compared to reference DevB (Fig. 4A). The SPR-data imply (i) a contact interface of DevB to DevAC involving both the lipoyl and the β-barrel domain, and it reflects (ii) a 171 stabilizing effect of DevB hexamers on the binding to DevAC (B-DαHD > B-PαHD, B-αHD > B- βBD, BxLipD; Fig. 4A). This behavior is similar to the binding of DevB to TolC (B-DαHD > B-βBD > B-PαHD, BxLipD > B-αHD; Fig. 2).