Figure S1. Figure S2.
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- Ingvild Eriksen
- 4 år siden
- Visninger:
Transkript
1 Figure S1. MALS-SEC trace of Tm14S receptor fragment (residues ). Rayleigh ratio (LS) and differential refractive index (RI) shown as a function of retention time. Due to the lack of Trp and Tyr residues Tm14S has a very small ε(280 nm) and as such UV absorption trace is not shown. Decrease in absolute MW estimate from ~40 kda to ~25 kda across the elution peak indicates an exchange between tetrameric and dimeric species (subunit MW = 9.9 kda). Figure S2. Electron density maps (A) Fo-Fc simulated-annealed omit electron density map of the Tm14S N-terminal helix in the region of P5 binding (blue contours at 3σ, red contours at 5 σ). (B) Fo1 Fo2 difference Fourier map calculated from amplitudes of the 3.5 Å resolution data and the 4.5 Å resolution data phased with the high-resolution model (blue contours +3σ, red contours -3σ). Difference peaks localize to the junction between the hairpins.
2 a Score = 42.4 bits (98), Expect = 3e-10. Identities = 36/137 (26%), Positives = 66/137 (48%), Gaps = 9/137 (7%) CheW1_Tmar 18 EFLVFTLGDEEYGIDILKVQEIRGYDQVTRIANTPAFIKGVTNLRGVIVPIVDLRIKFSQ 77 E L F + ++ D T + + F++GV NLRG I+P+V+L K CheW_Ecol 12 EVLSFEIDEQALAFDVDNIEMVIEKSDITPVPKSRHFVEGVINLRGRIIPVVNLA-KILG 70 CheW1_Tmar 78 VDVDYNDNTVVIVLNLGQRVVGIVVDGVSDVLSLTAEQI---RPAPEFAVTLSTEYLTGL D +IV VG +VD V VL +T Q+ + +F + GL CheW_Ecol 71 ISFDEQKMKSIIVARTKDVEVGFLVDRVLGVLRITENQLDLTNVSDKFG-----KKSKGL 125 CheW1_Tmar 135 GALGDRMLILVNIEKLL 151 R++I ++I+K++ CheW_Ecol 126 VKTDGRLIIYLDIDKII 142 b Score = 97.4 bits (241), Expect = 2e-30. Identities = 50/135 (37%), Positives = 84/135 (62%), Gaps = 0/135 (0%) CheW2_Tmar 22 FTLGDEEYGIDILKVQEIRGYDQVTRIANTPAFIKGVTNLRGVIVPIVDLRIKFSQVDVD 81 F LG++ +G+DI+KV+ I T +I+GV NLRG I+P+V+LR KF D++ CheW_Ecol 10 FKLGNQVFGVDIMKVESIVEVEKIVPVPETAEYIEGVMNLRGKIIPVVNLRKKFKMPDME 69 CheW2_Tmar 82 YNDNTVVIVLNLGQRVVGIVVDGVSDVLSLTAEQIRPAPEFAVTLSTEYLTGLGALGDRM 141 +IV + ++G +VD VS+VL+LT I P+ Y+ GL + D + CheW_Ecol 70 DKKRAKIIVSMVKNTLIGFLVDDVSEVLTLTESDIEQPPQNLAGKGKNYILGLAKVRDDI 129 CheW2_Tmar 142 LILVNIEKLLNSEEM 156 +I++NIE++L SEE+ CheW_Ecol 130 VIILNIEEVLTSEEL 144 Figure S3. Pairwise sequence alignments between E. coli CheW protein and CheW1 (a) and CheW2 (b) from T. maritima. Locus tag numbers are: b1887, E. coli CheW; TM0701, T. maritima CheW1; TM0718, T. maritima CheW2.
3 M M M 2D TM CTTTTTCCCEEEEEEEEETTEEEEEETTTEEEEECTTTTTTTTTTTTTEEEEEEETTE-EEEEETHHHHHTTTTTTTT TM MKTLADALKEFEVLSFEIDEQALAFDVDNIEMVIEKSDITPVPKSRHFVEGVINLRGR-IIPVVNLAKILGISFDEQK Tpet_ MKTLADALKEFEVLSFEIDEQALAFDVDNIEMVIEKSDITPVPKSRHFVEGVINLRGR-IIPVVNLAKILGISFDEQK Tnap_ MADALKEFEVLSFEIDEQALAFDVDNIEMVIEKSDITPVPKSRHFVEGVINLRGR-IIPVVNLAKILGISFDEQK TRQ2_ MADALKEFEVLSFEIDEQALAFDVDNIEMVIEKSDITPVPKSRHFVEGVINLRGR-IIPVVNLAKILGISFDEQK CTN_ MEVKKLPDMREFEVLSFEVEDQALAFDVDNIEMVIEKPEITPVPKSKHFVEGVINLRGR-IIPVVNLAKVLDISFDDSK Fnod_ MEEIREFEVLVFKALNQEMAIDVEMVEIVIDKTDITPVPKARKIIEGVINLRGK-IIPVISLPTLLAGSDSKEN Ferpe_ MNGLKGVNIVADNMEFEVLVFKALNQEMAIGVEMVEIVIEKTEITPVPRAKKIIEGVINLRGK-IVPVISLPMLLANESNTE- THA_ MNVETEFEVLSFNICNQEMSFDVDYVEIVIDKDEITPVPKSKEVIEGVINLRGR-IIPVVNLRKILGGICSNMD Tmel_ MSVETEFEVLSFNVCNQEMSFDVDFVEIVIDKDEITPVPKSKEVIEGVINLRGR-IIPVVNLKKILGGICQNID Tlet_ MANENEFEVFVFSIGEQEMAFDVDYVSMVIEKPEITPVPKSKKTVLGVMNLRGR-IVPVVSLNLLLDIESSDDL Theth_ MAEQKIFEVFVFSIGDQEMAFDIEYVSMVIDKAEITPVPRSKPTVLGVMNLRGR-IVPVISLPKLLGLQDSASE Marpi_ MQKEILSFEVVNQEFGIDVDYVEMVIEKEEITPVPNSRVFIEGVVNLRGR-IVPVFDLTKILDLEVPEDI Pmob_ MEDVLSFKIVDQEYALPIENIESVVDKTDVTPVPNSKYFVVGLINLRGR-IVPVIELTKILGIEVPEDH consensus...-lhsfph.pqthuhsl-.lp.vl-k.-ltpvppu+.hl.glhnlrg+ IlPVhpL.hlLs...p.. 2D Ecoli TTTTTTCCCTTTTCCEEEEEEEEETTEEEEEEHHHEEEEEETTTEECTTTTTTTTTEEEEETTE-EEEEEEHHHHHCCTTTTTT TM MKMELKVVTFKLGNQVFGVDIMKVESIVEVEKIVPVPETAEYIEGVMNLRGK-IIPVVNLRKKFKMPDMEDK Tnap_ MELKVVTFKLGNQVFGVDIMKVESIVEVERIVPVPETAEYIEGVMNLRGK-IIPVVNLRKKFKMPDIDDK Tpet_ MELKVVTFKLGNQVFGVDIMKVESIVEVERIVPVPETAEYIEGVMNLRGK-IIPVVNLRKKFKMPDIDDK TRQ2_ MELKVVTFKLGNQVFGVDIMKVESIVEVEKIVPVPETAEYIEGVMNLRGK-IIPVVNLRKKFKMSDMEDK CTN_ MELKVVTFKLGNQVFGVDIMKVESIVEVEKIVPVPETAEFIEGVMNLRGK-IIPVVNLRKKFKMPDMEDK Tlet_ MELKVVSFLLGEEKFALDIMSVDSIVEMGKIVKIPESADYVEGIMNLRGN-VIPIINLKKKFKMKITDRA Theth_ MELKVVSFLLGEEKFALDIMVIDSIVEVGKIVKIPESREYVEGVMNLRGN-VIPVINLKKKFKMKDVERK Fnod_ MELKFLTFHLDKEIFAINIMKVERVKEYEKTTKMPNIAEYVEGIINLMGE-IVPIINLRKKFMLPDFENK Ferpe_1035 MFSDCDIIKIAVNQKLGGESMELKFLTFYLGEEVFAINIMKVERVKEYEKTTKIPNIADFVEGIINLMGE-IVPIINLRKKFLMDDFANK THA_ MELKVLTFVLGDEEFAIDIMKVDRVKEYEKTTKLPNSKDYVEGIINLMGE-VIPIINLRKKFMLEDFEDK Tmel_ MELKVLTFMLGDEEFAIDIMKVDRVKEYEKTTKLPNSLDFVEGIINLMGE-VIPIINLRKKFMLQDFDEK Marpi_ MSVELKAVSFSVDDEKFAIDINHIDTVIEYQKTTKIPESSDFIEGIVNFRDG-VIPIINLRVKFNYPQFEDK Pmob_ MPSELKIVTFNIGKEKFGLDIMNVDAVIEYEETTKLPNASDYFEGVINYRNEEVLPIINLRRKFKMSDFEDR consensus...elkhlof.lspp.fulsi..l-tlhehtchs.hpp..-aheglhnhhst.llpllnl+hkf.h..htpt MM 2D TM TCEEEEEEET--TEEEEEEECEEEEEEEEETTTTTT-TTCCTTTT--TTTCEEEEETTEEEEEECCHHHH--HHHHHCCCC TM MKSIIVARTK--DVEVGFLVDRVLGVLRITENQLDL-TNVSDKFG--KKSKGLVKTDGRLIIYLDIDKII--EEITVKEGV Tpet_ MKSIIVARTK--DVEVGFLVDRVLGVLRITENQLDL-TNVSDKFG--KKSKGLVKTDGRLIIYLDIDKII--EEITVKEGV Tnap_ MKSIIVARTK--DVEVGFLVDRVLGVLRITENQLDL-TNVSDKFG--KKSKGLVKTDGRLIIYLDIDKII--EEITVKEGV TRQ2_ MKSIIVARTK--DVEVGFLVDRVLGVLRITENQLDL-TNVSDKFG--KKSKGLVKTDGRLIIYLDIDKII--EEITVKEGV CTN_ MKSIIVAKTK--DVEVGFLVDRVLGVLRITEEHVEM-VDVSDKFG--KKSKGLIKTDGRLIIYLNIDEII--EEITVKEGV Fnod_ QKKIVIVKIE--DVEFGLIVNEVVGVLRTNSSELETNLGKINTYG--KKAKGLIKKGTRLIVYLNLEEIL--REITGSEVA Ferpe_ YKKIIIAKVE--DVEFGLMVDEVVGVMRVRSEELETNLGKMNTYG--KKAKGLIKKDARLIVYLNLEEIV--KEIVGSEVA THA_1191 ENNFKKIIITKIR--DIEMGFLVENVKGVLRTSNSEIDRAFRDVDTYG--KKAKGLIKKGERLVVYLDIEEIL--NEIIGVEEV Tmel_0932 SVEFRKIIITKIR--DIEVGFLVENVKGVLRTSHEEIDQSFRDVDTYG--KKAKGLIKKGERLVVYLDIEEIL--NEIIGIEEV Tlet_0630 E--E-KVVVVNCE--DVEAGFIVNKVKGVLTCKVTEVEK-LQKLEKFG--EKSKGIIKKGEELIVYLNLEKTL--GELMAASEYSNEGVW Theth_0693 E--VGKIVVVNWE--EIEAGFAVSKVKGVTRCSIEEVEK-LYKLDKFG--EKCKGIIKKGDELIVYLSLDKIL--EELTAAYEHV----- Marpi_0716 V--FDSIIIMKHE--KEEIGILVNKVNNVISFDDEELDVLPAKSGKFV--GKVNGVLKINNRLIVYLNLEKLF--ESLETK Pmob_1391 I--YQNILVLKIN--EEEIGMYVDEVENVLSIDPEKLEKFQSKESVYS--DKVKGVIKIENRLIVYLDLESVL--EAELEK consensus...plllhphp c.ehghhvppv.svhphp.ppl-...thas tkspgllkhttclllylsl-phhtt... 2D Ecoli T--TTEEEEEEET--TEEEEEEECEEEEEEEEETTTEEBCCCCCCCCCC-HHHCCEEEEETTEEEEECCHHHHHHHHHHHHHTTTTTTCC Tnap_0516 K--KAKIIVSMVK--DTLIGFLVDDVSEVLTLTESDIEQPPQNLAGKG-KSYILGFAKVRDDIVIILNIEEVLTSEELVEISNINV-- Tpet_0211 K--KAKIIVSMVK--DTLIGFLVDDVSEVLTLTESDIEQPPQNLAGKG-KSYILGFAKVRDDIVIILNIEEVLTSEELVEISNINV-- TM0718 K--RAKIIVSMVK--NTLIGFLVDDVSEVLTLTESDIEQPPQNLAGKG-KNYILGLAKVRDDIVIILNIEEVLTSEELVEISNINV-- TRQ2_0209 K--KAKIIVSMVK--DTLVGFLVDDVSEVLTLTESDIEQPPQSLSGKG-KKYILGLAKVRDDIVIILNIEEVLTSEELVEISNINV-- CTN_1866 T--RAKIIVAMVK--DTQVGFLVDDVSEVLTLTEEDVEQPPQNLAGRG-REYILGLAKVRDDIVIILNIEEVLTSEELVEISNINV-- Tlet_0174 P--SSKIVVINLD--DRKIGLLVDQVHEVLTITDKQIEQPPADLGKTK-TNIVLGIAKFEEDLLIILNGKELLTSEEQIQLEGLTKLA Theth_0294 P--SAKIIVVNLD--DRRVGLLVDQVHEVLTLTDQQIEQPPADISGTK-SNLVLGIAKLGEELLIILNAKEILSTQEQIELVNLTSKA Fnod_0822 E--KTKIIVVKLE-NNKKVGFLVDDVREVLTVTEDKIDEPPAHIGGMSNVKFISGVIKLENEMILTLEVDNLLTSEEKLALANI---- Ferpe_1035 E--KSKIIVVKLE-NGKKVGLLVDDVREVLTVTEDMIDEPPAHVAGMSGAKFISGVIKLENEMVLTLEVDNLLTTEEKIALANIG--- THA_515 E--KSKIIVIRFEENGKKMGFLVDDVKEVITLSGDKIDQTPEYS-GLS-AEFLLGIAKLENRMILILDVEKVLKKEEKLAIENIIK-- Tmel_0275 E--KTKIIVIRFE-NEKKMGFLVDDVKEVITLSGDQIDQTPEYS-GES-SQFLLGVAKLEDRMILILDVEKILKKEEKIEISNMVK-- Marpi_0748 L--KAKVLVVKIE--DKKYGLMVDEVKEVMTITQEQIEEAPEVG-GTK-ANYITGIIKTKDSMIFLIDVEKILTEEEKIEIEKAIK-- Pmob_0388 S--HAKVIVLKID--QRRVGIMVDDVKNVRSIDPNLINEKPNIG-GMRGADFISGIARLEDGMLVILDIDKLLTEEEKVAIDEVINNconsensus...psKllV..hc..tp.hGhhVDpVppVhols.p.lpp.Pt...t.t..phl.Ghh+htpthlhhlphcplLpppE.ltl.th... Figure S4. Multiple sequence alignment of full-length CheW protein sequences from Thermotogae. CheW1 (TM0701) orthologs are on the top panel, CheW2 orthologs on the bottom panel. Consensus (100%) is shown underneath the alignment. 2D structure based on the 3D structure of TM0701 (PDB ID: 1K0S) and E.
4 coli CheW (PDB ID: 2HO9) are shown above the alignment. MCP-binding sites revealed in NMR studies (PMID: ) are marked (M). Correlated mutations are highlighted. W W W A WAAWWAW WW W TM0014/ IGETLENIRSIEKLIQNIMRIARETNILALNATIEAARAGEAGKGFMIVANEVQNLSNETNEVTKQIVEKAREILESSQRSLE TM1428/ VSNVTESARNIEEIVRTIQSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQKATEEISQILENIREGVERTNE TM0918/ LRELVDSAKTIGEIVDTISSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQRATEDIAKMLSSLRTTIEHVEN TM0429/ LRELVDSAKTIGEIVDTISSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQRATEDIAKMLSSLRTTIEHVEN TM1143/ VESFQKGAEEITSFVETINAIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQQASENVRRVVNEIRSIAEDAGK TM1146/ LNRLLSMAENINSIVDTINSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESKAATQQIGEILGKLRDEINNSSK TM0023/ LRELVDSAKTIGEIVDTISSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQRATEDIAKMLSSLRATIEHVEN TRQ2_0931/ IGETLENIRSIEKLIQNIMRIARETNILALNATIEAARAGEAGKGFMIVANEVQNLSNETNEVTKQIVEKAREILESSQRSLE TRQ2_0564/ VEELNEHARNIGQILDTINSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESKSATEKIGQILNQISQEVVKANE TRQ2_1321/ VSSVTESARNIEEIVRTIQSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQKATEEISQILENIREGVERTNE TRQ2_0009/ LRELVDSAKTIGEIVDTISSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQRATEDIAKMLSSLRATIEHVEN TRQ2_0515/ LRELVDSAKTIGEIVDTISSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQRATEDIAKMLSSLRATIEHVEN TRQ2_0922/ LRELVDSAKTIGEIVDTISSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQRATEDIAKMLSSLRATIEHVEN Tnap_0645/ IGETLENIRSIEKLIQNIMRIARETNILALNATIEAARAGEAGKGFMIVANEVQNLSNETNEVTKQIVEKAREILESSQRSLE Tnap_0162/ VEELNEHARNIGQILDTINSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESKSATEKIGQILNQISQEVVKANE Tnap_1383/ VSNVTESARNIEEIVRTIQNIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQRATEEISQILENIREGVEKTNE Tnap_0009/ LRELVDSAKTIGEIVDTISSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQRATEDIAKMLSSLRATIEHVEN Tnap_0654/ LRELVDSAKTIGEIVDTISSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQRATEDIAKMLSSLRATIEHVEN Tpet_0909/ IGETLENIRSIEKLIQNIMRIARETNILALNATIEAARAGEAGKGFMIVANEVQNLSNETNEVTKQIVEKAREILESSQRSLE Tpet_0550/ VEELNEHARNIGQILDTINSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESKSATEKIGQILNQISQEVVKANE Tpet_0009/ LRELVDSAKTIGEIVDTISSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQRATEDIAKMLSNLRTTIEHVEN Tpet_0900/ LKELVDSAKTIGEIVDTISSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQRATEDIAKMLSNLRTTIEHVEN Tpet_1365/ VSSVTESARNIEEIVRTIQSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQRATEEISQILENIREGVEKTNE CTN_0684/ IAETLENIKSIEELVRNIMKIARETNILALNATIEAARAGEAGKGFAVVASEVQNLSSETNRVTKQIEAKTREIIESTQRSLE CTN_1554/ VDRLSESAKNIGEIVETINSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESRNATQNITEILKGIMEESLQAKS CTN_1065/ VLKVTESARNIEEIVRTIQSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQRATEEISQILENIREGVERTNE CTN_1657/ LKELVESAKTIGEIVNTISSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQKATEDIAKMLSSLRGTIGHVEE CTN_0672/ LKELVESAKTIGEIVNTISSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESQKATEDIAKMLSSLRGTIGHVEE Tlet_0138/ VMELSNKAKNIGEIVETINSIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESKSATDKIGQMLTQIQQGAQQAST Tlet_1737/ VGGLTEDAKNIATIVETINSIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEQSKSATENIANILRKIQQSAKDADY Tlet_1846/ VEELSNSVKDINEIVNAINLIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEQSKAATERISQMLTDIHHKTVKARD Tlet_0651/ VQELVDLSNRIGEITDVIYNIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESRKATQEVADTLKKIKSQTELVAK Theth_1583/ TSKTILRFSDIQAMVKDIEKIAKQTNLLALNASIESARAGEYGRGFAVVAAEIQKLANESNLITKRITDEMNNLAVAVGESAK Theth_1487/ VSKLSENAKNIGKIVETINSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESKQATQNIENILKGIFEESMMAGK Theth_0350/ VLELAENAKNIGQIVETINSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESKNATQRISEILSQIQQSAQKASG Theth_1144/ VLELAESAKNIGQIVETINSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESKNATQKISEILSQIQQSAQKASM Theth_1708/ LRLLVEDAKNIGQIVETINSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESKNATQKISSILGRIHDSAEVAAK Theth_0502/ VKKLAENAQNIGEIVEAINSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESKQATGRIANILKQIQDQTREVST Theth_1214/ MEKLVQMSQRIETITNTISNIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESRRSTQSISETLKEIRDLTAVVAK Theth_1359/ LKELLESTNVIGNVVNTIESIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEQTQDATKNISKILGSLRDSVANVES FerpeDRAFT_1419/ TTQTLEMFSKVEHMVLEITKIAKQTNLLALNASIEAARAGEFGKGFAVVASEVQKLAGESNRVAKEINDLVKELSASVSEALN FerpeDRAFT_1507/ VEKLSESAKNIGEIVDTINSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESKQATQNIAEILRGIVDNSMKASE FerpeDRAFT_1716/ IVELTKFSNQIGEIVDTISSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESRQATKKIAEILGKIGEQAKRVEK FerpeDRAFT_0693/ IRDLSSATKNIGSIIETVRQIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEQTKTSTDTINSTISAVDTYSKALES Fnod_0355/ TTKTLEMFNNIENMVNEITKIAKQTNLLALNASIEAARAGEFGRGFAVVASEVQKLANESNKVAKDITNQVKELSKSVSDALE Fnod_0128/ IVELTKFSNQIGEIVDTISSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESRQATKKIAEILSKIGEQAKKVEK Fnod_1100/ VESFNRGAEEISGFVETINAIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESKRASDNVRRVVEEIKGIAENANK Fnod_1300/ INQLTEATKNIGDIIQSVRNIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEETKRSTDTITSTIETIRTYSKVLNE THA_162/ TSETFEKFSKVEELVKAIQRIAKQTNLLALNASIEAARAGEYGKGFSVVASEIQKLAEESQETSANISKIISEISNSVNESIN THA_537/ VDDLASNANNIGEIVETISSIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESKSATDKITQILKNIQQRSEDVRT THA_1159/ VENLSKRAKNINEIVETINSIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESKVATENISKILNEILKESINASQ THA_92/ VEEMESSSNMIEEIVGTISSIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESKNATQKISEILTTIKDQAFNVAK THA_1016/ VEEMESSSNMIEEIVGTISSIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESKNATQKISEILTTIKDQAFNVAK THA_263/ ISKLLDYTQNIGEIVNTIRNIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEETKGATDVITNTIQEITKSSDVIKD Tmel_1867/ TSKTFENFEVIKDLVKNIQKIAKQTNLLALNASIEAARAGEYGKGFSVVASEIQKLAEESQNTSKDISKIVGEISESVRTSLE Tmel_0322/ VDLLSDNAKNIGEIVETISSIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESKIATDKITSILKNIQNGSEAVRK Tmel_1579/ VNELSVSAGNIKDILETINQISEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESKNATENIADILEKIREEADKVNE Tmel_0749/ VNSLSTKAQNIEEIVETINSIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESKNATQKINEILVSIKDSSLKASS Tmel_1120/ VREMEESSTKIEEIVETISSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESKSQTQKIAEVLNAIKNQAVNVAK Tmel_1776/ VREMEESSTKIEEIVETISSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESKSQTQKIAEVLNAIKNQAVNVAK Tmel_0346/ SELLEKSVNAIGEILNVISGVAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEETKKSADEIAVKLGDISHRSKETAN Tmel_1816/ VESFSKGAQDITQFIETINQIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESKIASEKIKVVVDEIGKIAADARN Tmel_0144/ MKELSEYSKNIEQILETITSIADQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESRNATESIGKILTSIGKNISSSVK Tmel_0046/ IEDFLLSTKDIENIVLTIRDIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEETKNATNTITETIMKLDEKTRIINS Pmob_0699/ VSHLMDNFQEIEELTNVIKNVAKQTNILSINASIEAARAKEAGRGFAVVAEEIKKLSSETNIASNKISERIVNLSNQVLEVKG Pmob_0696/ VKALIERSENIGKIVNTINSITEQTNLLALNAAIEAARAGEAGKGFAVVADEIRSLAEDSKNSTSQIERMIEGIKNSTNVVND Pmob_0861/ VSKLLEKAENIGNIIQTINSITEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESGKATDEIGNILEDLKKSTNNVND Pmob_0228/ VETLASNAKNVQSIVETINSITEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESRNATDEISEILTNITQGTNKVNE Pmob_0509/ VETLASNAKNVQSIVETINSITEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESRNATDEISEILTNITQGTNKVNE Pmob_0496/ VETLASNAKNVQSIVETINSITEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESRNATDEISEILTNITQGTNKVNE Pmob_0137/ VETLASNAKNVQSIVETINSITEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESRNATDEISEILTNITQGTNKVNE
5 Pmob_1607/ VETLASNAKNVQSIVETINSITEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESRNATDEISEILTNITQGTNKVNE Pmob_0837/ VETLANNAKNVQSIVETINSITEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESRNATDEISEILTNITQGTNKVNE Pmob_1507/ SEILSKNSEEIKEIINTIDSITEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEESKKATENIAKIITDLIVISQKTNL Marpi_0631/ VSEALSSFDEVMEITKNINKIARKTNMLSINASIEAAKAKEHGRGFAVVAEEIQKLSQETNESAKQINEKINYLSQEIKNVLE Marpi_1925/ VKVLASETANIQEIVETINSITEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESKKATEEIANILGKIQNSASEVDK Marpi_1927/ VKVLASETANIQEIVETINSITEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESKKATEEIANILGKIQNSASEVDK Marpi_0554/ VKSLQEKSANIGEIVDTIISIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEETRKATENISDLLKGIKDEADMVSK Marpi_1926/ VKNLQEKSANIGEIVDTIISIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEETRNATENISDLLKGIKDEADAVSQ Marpi_1452/ VAVLAQKAKNIGEIVDTITNITEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESKKATEQIAVILNEIRNGAAEANK Marpi_1522/ VAVLAEKAQNIGEIVETITSITEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESRKATEEIAKILNEIKDGAAKANQ Marpi_0098/ VETLSEKAENIGEIVDTITTITEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESKNATEEIAKILTEIKNGTIQANK Marpi_0788/ VEELLKLAENIGEIVETINTITEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESKKATEEIAQILSHVQNGVKDVNG Marpi_1529/ VQALSEKAQNIGNIVETITKITEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEQSKSATEEIAKILISVKEGAQDANE Marpi_1789/ VSDLAGKAQNIGEIVETIGSITEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESRNATDKIADILKEIQESANTANE Marpi_1837/ VQNLANKAQNIEQIVETIENITEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESKRATEQIAEILGEIKDGADDASD Marpi_1293/ VKAVSDKAQIINEVVETITSISEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEESRNAAESIRENLGEVMTGVNEASD Marpi_1294/ VKNVSNKAQIINDVVETITSISEQTNLLALNAAIEAARAGEAGKGFSIVAEEIRKLAEESRSAAESIRENLGEVMLGVKETSD Marpi_0747/ IIKLFDKSKEIEDIVGKITEISKQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLADESNLSANSITENLKELVDDASEALE Marpi_1052/ VREFNDSATMINDFVDTVSNIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRVLAEESRKAADEIKEVVNGIINVSNDALD Marpi_1139/ IKELENYASNITEVIEQVSNIAEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEETRKATDEIAGILGSIKANVDKSTK Marpi_0832/ LDKLARFTENINEIIITINNITEQTNLLALNAAIEAARAGEAGKGFAVVADEIRKLAEQSKTSTEQISEILESLVIEVKSSVE consensus/50% VccLs-sA+NIsEIV-TIsSIAEQTNLLALNAAIEAARAGEAGRGFAVVADEIRKLAEES+pAT-cIucILssIpssspcssc consensus/60% lpcls-sacsit-iv-tissiaeqtnllalnaaieaarageag+gfavvadeirklaees+pat-ciucllspippsspcssc consensus/70% lppl.cpupsitcivctissiaeqtnllalnaaieaarageag+gfavvadeirklaeesppatcpiuphlpplpptsppspp consensus/80% lpph.ppsppitpllpoipsiseqtnllalnaaieaarageag+gfavvadeirklaeespputppisphlppltptstpstp consensus/90% ltth.p..ppitpllpsippiscqtnllalnauieaarageag+gfavvaseipklapeoppsoppisphlttl.tt..ts.p consensus/100%...h...t.l.thh..l..lscptnhlulnasieua+atehg+gf.lvatelp.lutpot..st.l...h..l... heptad number N06 N05 N04 N03 N02 N01 C01 C02 C03 C04 C05 C06 abcdefgabcdefgabcdefgabcdefgabcdefgabcde gabcdefgabcdefgabcdefgabcdefgabcdefgabcdef Figure S5. Multiple sequence alignment of the signaling domains from 92 MCPs from Thermotogae that were assigned to heptad classes. Aligned region corresponds to the TM0014 construct in the new structure. T36H MCPs are shown in bold, MCPs are in regular font. CheW (W) and CheA (A) binding sites identified by NMR studies (PMID: and ) are shown above the alignment. Class-specific residues are highlighted in grey. Position Ile135 shows correlated mutation pattern with positions Thr565 and Ile566 in the P5 domain of CheA (see Supplementary Figure 4). Consensus at varying stringency and N-terminal (N) and C- terminal (C) heptad numbering are shown underneath of alignment.
6 TM0702 TRQ2_0226 Tnap_0499 Tpet_0228 CTN_1883 Theth_0692 Tlet_0631 Ferpe_0477 Fnod_0690 THA_1190 Tmel_0933 Marpi_0715 Pmob_1390 consensus 80% consensus 100% M M 540 TLAIIQALLVKVNNLVYAIPIANIDTILSISKEDIQRVQDRDVIVIRGEVIPVYRLWEVLQIEHKEEL 540 TLAIIQALLVKVNNLVYAIPIANIDTILSVSKEDIQRVQDRDVIVIRGEVIPVYRLWEVLQIEHKEEL 540 TLAIIQALLVKVNNLVYAIPIANIDTILSISKEDIQRVQDRDVIVIRGEVIPVYRLWEVLQIEHTKEL 540 TLAIIQALLVKVNNLVYAIPIANIDTILSISKKDIQRIQDRDVIVIRGEVVPVYRLWEVLQIEHTKEL 540 TLAIIQALLVKVNNLVYAIPIANIDTTLRISRGDLQRVQDKNVIVIRGEVIPVYKLWEVLQIEHEREE 543 TLAIIQALLVKVGKYVYAIPIANIDSTLNIDRSQIKKIQDREVTVIRGEIVPIVKLWEVLGIPHELQD 527 TLAIIQALLVKVNKYVYAIPIANIDSTLNISKEQIQRIQDRQVTVIRGEIIPIAKLWDVLELPHDKEN 547 TLAIIQALLVKVNNYVYAIPISIIDSTLIISPEEIKVVQNEEVIVIRGEVIPLIKLWHVFNFPHEETP 541 TLAIIQALLVKVNNYVYAIPISIIDSTLIISSEDIKVVQNEEVIVIRGEVIPIIKLWEVFGMEHEQNP 548 TLAIIQALLVKVDKYIYAIPISVIDSTLIVLPDEIQMVQNKEVIVKRGEVIPIIKLWDVLGMKHNEEL 542 TLAIIQALLVKVNNLIYAIPISVIDSTLIVLPNEIQMVQNKEVIVRRGEVIPIIKLWDVLNLEHSNEF 558 TLSIIQVLLVTLDDYVYAIPIANIDTTQKITDGEIRIVQGNEVFVLRGEIIPLIRLRELFKMPPKEED 542 TLAIIEALLITVNEQVYAIPIANIDTTQRLSDGELKIVQGQEVFLLRGEVMPVVRLRKLFGYEKKKDS TLAIIQALLVKVsphVYAIPIusIDohL.ls.t-IphlQsc-VhVlRGEllPlh+LW-Vhth.Hppp. TLuIIpsLLlplsp.lYAIPIu.IDoh..l..tplphlQsppVhlhRGElhPlh+Lhclhth..p.p. TM0702 EEMEAVIVRVGNRKYGIVVDDLLGQDDIVIKSLGKVFSEVKEFSGAAILGDGSIALIINVSGIV TRQ2_0226 EEMEAVIVRVGNRKYGIVVDDLLGQDDIVIKSLGKVFSEVKEFSGAAILGDGSIALIINVSGIV Tnap_0499 KEMEAVIVRVGNRKYGIVVDDLLGQDDIVIKSLGKVFSEVKEFSGAAILGDGSIALIINVSGIV Tpet_0228 KEMEAVIVRVGNRKYGIVVDELLGQDDIVIKSLGKVFSEVKEFSGAAILGDGSIALIINVSGIV CTN_1883 EEMEAVIVRIGNRKYGIIVDELLGQDDIVIKSLGKVFSDVREFSGAAILGDGSIALIINVSGIV Theth_0692 GFYNTVIVRSGNRKIGIAVDTLIGQEDIVIKSLGKIFSDVKLFSGGAILGDGSIALILDVTNIA Tlet_0631 GTYNTVIVRIGNRKYGLAVDTLIGQEDIVIKSLGKIFSDVRIFSGGAILGDGSIALILDVANIV Ferpe_0477 SEMKVVVVKHGNRKYGLTVDTLIGQEDIVIKSLGKIFSDVKIFSGGATLGDGSIALILDVANIVEMA 681 Fnod_0690 HEMNVVVVKSGAKKYGLAVDTLIGQEDIVIKSLGKVFSDVKIFSGGATLGDGSIALILDITNIVE THA_1190 DELNVVVVKVANRKYGIAVDTLIGQEDIVIKSLGKLFADVKEFSGGATLGDGSIALIIDTLNLVE Tmel_0933 DELNVVVVKVANRKYGIAVDSLIGQEDIVIKSLGKLFTDVKEFSGGATLGDGSIALIIDTLNLVE Marpi_0715 FVEKIVIVKTANRKYGVVVDNLLGQDDIVIKSLGKYLNSVKEFSGGAILGDGRIALILDITNLNEQ- 691 Pmob_1390 SSEYIIIVKLGNKKYGVVVDRLLGQDDIVIKSLGSLLKDVKEFSGGAILGDGRIALILDVASLI consensus 80% tphpsvlv+hunrkyglsvdpllgq-divikslgklfs-vk.fsguahlgdgsialilslssll... consensus 100%...hllV+.us+KhGlhVDpLlGQ-DIVIKSLGphhtpV+.FSGuAhLGDGpIALIlsh.sl... Figure S6. Multiple sequence alignment of P5 (CheW-like) domains from CheA protein sequences from Thermotogae. Residues identified as contact sites for MCP interaction by NMR studies (PMID: ) are marked (M). Residues predicted to be contact sites for interacting with MCP are highlighted: invariably conserved residues are in black; residues showing correlated mutation pattern with corresponding contact residues in MCP are highlighted in yellow and blue. Consensus (80 and 100%) is shown underneath of alignment. Figure S7. A pair of allele-specific repressor mutations between Tsr and E.coli CheW (w98; r115; yellow bonds) map in close proximity on at the upper interface between CheW (green) and the end of Tm14 S (pink).
7 Organism Genome Status CheA CheW W1/W2 MCP Heptad Class Thermotoga maritima MSB8 Complete TM0702 TM0701 W1 TM0014 T36H TM0718 W2 TM0918 TM0023 TM0429 TM1428 TM1143 TM1146 Thermotoga neapolitana DSM 4359 Complete CTN_1883 CTN_1884 W1 CTN_0684 T36H CTN_1866 W2 CTN_1657 CTN_0672 CTN_1554 CTN_1065 Thermotoga petrophila RKU- 1 Complete Tpet_0228 Tpet_0229 W1 Tpet_0909 T36H Tpet_0211 W2 Tpet_0009 Tpet_0900 Tpet_0550 Tpet_1365 Tpet_1766 UNCAT Thermotoga sp. RQ2 Complete TRQ2_0226 TRQ2_0227 W1 TRQ2_0931 T36H TRQ2_0209 W2 TRQ2_0009 TRQ2_0922 TRQ2_0515 TRQ2_0564 TRQ2_1321 Thermotoga naphthophila RKU- 10 Complete Tnap_0499 Tnap_0498 W1 Tnap_0645 T36H Tnap_0516 W2 Tnap_0009 Tnap_0654 Tnap_0162 Tnap_1383 Thermotoga thermarum DSM 5069 Complete Theth_0692 Theth_0693 W1 Theth_1583 T36H Theth_0294 W2 Theth_1359 Theth_0350 Theth_1487 Theth_1214 Theth_0502 Theth_1144 Theth_1708
8 Theth_0562 UNCAT Theth_0351 UNCAT Thermotoga lettingae TMO Complete Tlet_0631 Tlet_0630 W1 Tlet_0138 Tlet_0174 W2 Tlet_1737 Tlet_1846 Tlet_0651 Tlet_0941 UNCAT Tlet_0858 UNCAT Thermosipho africanus TCF52B Complete THA_1190 THA_1191 W1 THA_162 T36H THA_515 W2 THA_537 THA_1159 THA_1016 THA_92 THA_958 THA_263 THA_1009 UNCAT THA_841 UNCAT THA_1449 UNCAT Thermosipho melanesiensis BI429 Complete Tmel_0933 Tmel_0932 W1 Tmel_1867 T36H Tmel_0275 W2 Tmel_0749 Tmel_0322 Tmel_1120 Tmel_1776 Tmel_1579 Tmel_1816 Tmel_0144 Tmel_0851 Tmel_0046 Tmel_0416 UNCAT Tmel_0451 UNCAT Tmel_0346 UNCAT Tmel_1144 UNCAT Fervidobacterium nodosum Rt17- B1 Complete Fnod_0690 Fnod_0691 W1 Fnod_0355 T36H Fnod_0822 W2 Fnod_0128 Fnod_1100 Fnod_1300 Fnod_0170 UNCAT Fnod_0912 UNCAT Fnod_0757 UNCAT Fnod_1435 UNCAT Fervidobacterium pennivorans DSM 9078 Draft v1 FerpeDRAFT_1376 FerpeDRAFT_1375 W1 FerpeDRAFT_1419 T36H FerpeDRAFT_0969 W2 FerpeDRAFT_1507 FerpeDRAFT_1716 FerpeDRAFT_1552 FerpeDRAFT_0693
9 FerpeDRAFT_1433 UNCAT FerpeDRAFT_0572 UNCAT Petrotoga mobilis SJ95 Complete Pmob_1390 Pmob_1391 W1 Pmob_0699 T36H Pmob_0388 W2 Pmob_0696 Pmob_0861 Pmob_1607 Pmob_0228 Pmob_0496 Pmob_0509 Pmob_0837 Pmob_0137 Pmob_1633 UNCAT Pmob_1507 UNCAT Marinitoga piezophila KA3 Complete Marpi_0715 Marpi_0716 W1 Marpi_0631 T36H Marpi_0748 W2 Marpi_1837 Marpi_1493? (HGT) Marpi_1925 Marpi_1927 Marpi_0554 Marpi_1926 Marpi_1052 Marpi_0832 Marpi_1757 Marpi_1789 Marpi_0098 Marpi_1522 Marpi_0747 Marpi_1452 Marpi_1132 UNCAT Marpi_1139 UNCAT Marpi_0788 UNCAT Marpi_1293 UNCAT Marpi_1294 UNCAT Marpi_1492 UNCAT Marpi_1529 UNCAT Marpi_0896 UNCAT Kosmotoga olearia TBF Complete - - n/a - n/a Thermotogales bacterium mesg1.ag.4.2 Draft v1 - - n/a - n/a Table S1. The list of CheA, CheW and MCP protein sequences from Thermotogae genomes. Any MCP that did was classified as T36H or was labeled uncategorized (UNCAT) and excluded from analysis due to non-uniform distribution.
10 Interaction MCP residue CheW residue CheA residue C β Distance (Å) 3UR1 MCP:CheW I142 G (Bottom) I142 V R146 E R146 V Unzipped MCP:CheW (Top) 3UR1 MCP:P5 (Top) Unzipped MCP:P5 (Bottom) R146 V G110 E G110 V G110 V E114 V E114 I R117 T R117 I S118 I S118 Q K121 I I135 I L138 I N139 T N139 I T141 Q I142 I I142 I Table S2: Contact sites in MCP-CheW and MCP-CheA interactions prospected using 6 Å beta carbon cutoff dependent protein scanning.
11 MCP residue CheW interaction CheA interaction Arg131 Glu132 Ile135 Ala137 Leu138 Asn139 Ala140 Thr141 Ile142 Glu143 Ala145 Arg146 Ile156 Asp159 Glu162 Table S3. MCP residues interacting with CheW and P5 revealed by three independent approaches: (1) Bottom-up bioinformatics, (2) Structure/ Top- down bioinformatics, (3) NMR (data from PMIDs: and ). Residues identified by more than one method are highlighted. CheW residue MCP interaction Glu12 Leu14 Val27 Asp28 Ile30 Lys36 Asp38 Val98 Leu99 Gly100 Val101 Lys121 Table S4. CheW residues interacting with MCPs revealed by three independent approaches: (1) Bottom-up bioinformatics, (2) Structure/ top down bioinformatics, (3) NMR (data from PMIDs: and ). Residues identified by more than one method are highlighted.
12 CheA residue MCP interaction 1 2 Ile563 Thr565 Ile566 Gln563 Table S5. CheA residues interacting with MCPs revealed by two independent approaches: (1) Structure/ top down bioinformatics, (2) NMR (data from PMIDs: and ). Residues identified by more than one method are highlighted.
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