Residues involved in metal binding are highlighted in red, the consensus motif E(D)TG. (PRS and ETQ .... 472324017 P.denitrificans ATCC RLIMOS TTV HDALA. ..... observed only for the sample (red) and not in the controls (green and blue).
Supplementary figures
FIGURE S1
PA4781 semi-Apo +/- divalent metals
PA4781 fully-Apo +/- divalent metals
-1
semi-Apo
2
Mean Residue Ellipticity (deg cm dmol )
5000 Zn Ni Mn
0
fully-Apo Zn Ni Mn Co
Co Cu
-5000
Fe
-1 10
4
4
-1.5 10
-2 10
4
4
-2.5 10
210
220
230
240
250
210
220
230
240
250
Wavelength (nm)
Wavelength (nm)
Figure S1. Circular Dichroism (CD) analysis. CD spectra, indicative of the secondary structure profile, of the semi-Apo (containing Ni2+ in the sole high affinity site) and of the fully-Apo PA4781 incubated with different divalent metals (metal/protein molar ratio = 4). In both cases the CD spectra in the presence of metals, including Zn2+, superposes with that of the semi-Apo or fully-Apo samples, indicating that addition of tested metals does not affect folding.
1
FIGURE S2
Figure S2. Sequence alignment between PA4781 and PmGH HD-GYP domains. Residues involved in metal binding are highlighted in red, the consensus motif E(D)TG (PRS and ETQ in PA4781 and PmGH, respectively) is boxed in yellow. Notice the two long insertions at the level of Loop-1/2 and Loop-3/4 that further distinguish PA4781 from PmGH. Also notice that the GYP-Loop displays the highest degree of conservation (70% identity).
2
FIGURE S3 PA4781
PmGH
15599975_P.aeruginosa_PAO1 160
15599975_P.aeruginosa_PAO1 152988376_P.aeruginosa_PA7 472324017_P.denitrificans_ATCC 516091093_P._nitroreducens 575528596_Pseudomonas_sp._M1 544802557_P.alcaligenes 517531475_A.acidaminophila 288940945_A.vinosum_DSM_180 34496338_C.violaceum_ATCC_1247 493140715_C.basilensis 372487460_D.suillum_PS 498144707_A.radicis 407939109_Acidovorax_sp. 71909165_D.aromatica_RCB 517819128_A.toluclasticus 522196548_O.bacterium_AB_14 515524292_C.agarivorans 34497952_C.violaceum_ATCC_1247 491656777_V.mimicus 496733920_Vibrio_sp._RC586 491493257_A.diversa 549689089_V.nigripulchritudo 498066214_P.rubra 518476831_N.itersonii 511824815_A.albus 225850538_P.marina_EX-H1 495562704_Hydrogenivirga_sp. 551219240_Desulfurobacterium_s 489051231_P.haloplanktis 495447713_Pseudoalteromonas_sp 567931853_P._sp._NW_4327 495388329_Pseudoalteromonas_sp 77360093_P.haloplanktis 515078141_P.haloplanktis 493845294_A.bacterium_TW-7 584421140_P.lipolytica 519054894_G.chinensis 551324596_P.ruthenica 496119206_G.lipolytica 498247266_P.spongiae 119776243_S.amazonensis_SB2B 519026623_Methylotenera_sp._1P 490545238_V.orientalis 490886322_V.tubiashii 497412599_M.lonarensis 515633726_V.crassostreae 516227747_Vibrio_sp._624788 515642306_V.splendidus 430749652_T.composti_KWC4 516366640_Bacillus_sp._ZYK 551223028_Geovibrio_sp._L21 526461586_A.butzleri_7h1h 522074828_D.curvatus 495614505_S.gotlandica 78486250_T.crunogena_XCL 83312538_M.magneticum_AMB-1 550991115_C.Symbiobacter_mobil 242281256_D.salexigens_DSM_263
1
PA4781
PmGH
5
PmGH
10
190
200
20
25
30
35
40
. 250
15599975_P.aeruginosa_PAO1 152988376_P.aeruginosa_PA7 472324017_P.denitrificans_ATCC 516091093_P._nitroreducens 575528596_Pseudomonas_sp._M1 544802557_P.alcaligenes 517531475_A.acidaminophila 288940945_A.vinosum_DSM_180 34496338_C.violaceum_ATCC_1247 493140715_C.basilensis 372487460_D.suillum_PS 498144707_A.radicis 407939109_Acidovorax_sp. 71909165_D.aromatica_RCB 517819128_A.toluclasticus 522196548_O.bacterium_AB_14 515524292_C.agarivorans 34497952_C.violaceum_ATCC_1247 491656777_V.mimicus 496733920_Vibrio_sp._RC586 491493257_A.diversa 549689089_V.nigripulchritudo 498066214_P.rubra 518476831_N.itersonii 511824815_A.albus 225850538_P.marina_EX-H1 495562704_Hydrogenivirga_sp. 551219240_Desulfurobacterium_s 489051231_P.haloplanktis 495447713_Pseudoalteromonas_sp 567931853_P._sp._NW_4327 495388329_Pseudoalteromonas_sp 77360093_P.haloplanktis 515078141_P.haloplanktis 493845294_A.bacterium_TW-7 584421140_P.lipolytica 519054894_G.chinensis 551324596_P.ruthenica 496119206_G.lipolytica 498247266_P.spongiae 119776243_S.amazonensis_SB2B 519026623_Methylotenera_sp._1P 490545238_V.orientalis 490886322_V.tubiashii 497412599_M.lonarensis 515633726_V.crassostreae 516227747_Vibrio_sp._624788 515642306_V.splendidus 430749652_T.composti_KWC4 516366640_Bacillus_sp._ZYK 551223028_Geovibrio_sp._L21 526461586_A.butzleri_7h1h 522074828_D.curvatus 495614505_S.gotlandica 78486250_T.crunogena_XCL 83312538_M.magneticum_AMB-1 550991115_C.Symbiobacter_mobil 242281256_D.salexigens_DSM_263
180
15
15599975_P.aeruginosa_PAO1
210
220
230
240
45
50
55
60
65
70
75
80
85
90
TT
260
270
280
290
300
310
320
330
TALLQGHTRAGRDALA.SAERRLG.QPSGFLRFARQIAYSHHERWDGRGFPEGLAGERIPLAARIVALADRYDELTSRHAYRPPLAHAEA TALLQAHTRVGRDALA.DAERRLG.QPSGFLRFARQIAYSHHERWDGRGFPEGLAGERIPLAARIVAIADRYDELTSRHAYRTPLAHAEA RLIMQSHTTVGHDALA.AAERKLG.YPADFLRYAKEIAYSHHERWDGGGYPEGLAGERIPMAARMLTLIDCYDELTSRHAYRTSLGPDEA RLILESHTTVGRDALA.AAERKLG.SPADFLRFAKEIAYSHHEHWDGSGFPEGLAGEQIPLGARMLSLIDCYDEYTCRHAYRSSLTPEEA QLLMQSHTRIGHDALA.AAERRLG.SPAEFLRYAKEIAYGHHERWDGSGYPRGLRGEQIPLSARLLALIDCYDELTSRHPYHATLAPDEA LRLMQSHTRLGREALE.RAEARLGGSEQSFLGYAKEIQYGHHERWDGSGYPQGLRGDQIPLAARLMAVVDHYDGLTSYHPYRSSLSGDAA FSLVKRHAQAGRKSIE.EAGEFLG.VPEPFLRFAKEMACYHHERWDGNGYPQGLAGERIPVSARLMALADVYDAIVSRRVYKEPALHEDA WEIMKTHAKLGRDAIE.QAERDVD.RPLEFLVMAKDIAHYHHERWDGKGYPEGLAGDDIPIAARLMALADVFDALISQRVYKPPMSFEEA FAIMKQHAALGRDAIV.AAERQLG.LEVAFLKFAKEIACSHHEKWDGSGYPFGLKGDEIPVAGRLMALADVYDALINRRIYKPPMPHAQA FEVMKTHTTLGRDAIA.HAEAQCG.TPNSFLRYAREIAHYHQEKWDGSGYPDGLSGDAIPVSARLMAIADVYDALISRRVYKPAFPHEQA FEIMKTHTTLGRDAIL.HAEVRLN.SPNTFLRFARDIAYAHQEKWDGSGYPLGLRGDEIPVAARLMAVADVYDALISRRVYKPPFPHEQA FEIMKTHTTLGRDAIV.AAETETT.QNNPFFRYAKEIAYSHQEKWDGSGYPEGLVGNTIPLSARLMAVADVYDALISERVYKPAFTHEHA FEIMKTHTTLGRDAII.AAETGTT.QDNPFFRYAKEIAYSHQEKWDGSGYPEGLVGNTIPLSARLMAVADVYDALISERVYKPAYSHEQA FELMKAHTTLGRDAII.NAERRLG.ISVDFLNHAKEIAYSHQEKWDGSGYPEGLAGDAIPISARIMAVADVYDALISRRPYKNPFPHDKA FETMKAHTTLGRDAII.HAERKLG.MDVDFLRLAKEIAYYHQEKWDGSGYPEGIAGDAIPISARLMALADVYDALISRRVYKEGMSHEEA FEIMKTHTTLGRDAIR.QAERELG.LEVDFLKFAKEIAYGHQEKWDGSGYPEGLSGDNIPISARLMAVADVYDALISRRVYKAGMSHEAA FEIMKTHTTLGRDAII.QAEEHLG.VQLPFLTFAKEIAYGHQEKWDGSGYPEGLSGDDIPISARLMAVADVYDALISRRVYKDPMPHDKA FEVMKTHTVLGRDTIA.SAERMLD.APSSFLRLAREIAYCHQEKWDGSGYPQGLAGEAIPLSARLMAVADVYDALISRRVYKPPFSHEKA FEIMKLHTVLGRDVID.TVEQCIH.FECDFLTFAKEIAYSHQEKWDGSGYPEGLKGAEIPLSARLMALADVYDALISKRVYKPPFSHEKA FEIMKSHTVLGRDVID.AVEECIH.FECDFLTFAKEIAYSHQEKWDGSGYPEGLKGEEIPLSARLMALVDVYDALISRRVYKPPFSHEKA FEVMKSHTTQGLKALK.AVEEALD.FECAFLNLANEIAYSHQEKWDGSGYPEGLKGEAIPLSARLMSVADVYDALISKRVYKPPFSHEKA FDIMKGHSKLGGDTID.AVEKTLG.VTCAFLQHAKDIAYYHQEKWDGSGYPDHLVGEDIPLSARLMAVADVYDALISSRCYKPPFPHSKA FEIMKSHTVFGRNAIE.TAESALG.QTDSFLQTAKEISHYHHEKWDGSGYPEGLKGEDIPLSARLMAIADVYDALISRRVYKDAMEHSDA MEVMKRHAELGQQALT.AALEEQAGSPSDFLRYAVEISHYHHEKWDGSGYPHGLSGRDIPLSARLMAVADVYDALISRRVYKPPFPHEKA WQIMRQHPEIGADALA.EAERQFGSEDAAFLQYAKEIALSHHEKWDGSGYPEGLAGNDIPLSARLMALADVYDALISKRVYKPAFSHDKA WEIMKKHTIYGYEILK.G.G......DSRLLQIAADIAIEHHERWDGTGYPFGKKGEEISIYGRMTSISDVFDALTSDRPYKKAWDMDRT WDVMKKHTIIGYEILK.D.S......SSELLQMAALVALDHHERWDGTGYPNGKKGEEISLWGRITSVADVFDALLSKRPYKEPWSLERT RKIMEKHTIIGFNILK.G.S......KSRLLQIAALIALEHHEKFDGTGYPYGKKGEEISIYGRIAAAVDVFDALISKRPYKPAWQKEKV FDHMKEHATIGAQILA.N.S......SSPLLQLAHTLAIEHHEKWDGSGYPNGIKGEEISVEGRIVAIADVFDALTSKRPYKEAWSVEKT FTYMKEHAVIGAKILA.N.S......SSPLLQLAHKLAIEHHEKWDGSGYPNGIKGEEISIEGRIVAVADVFDALTSKRPYKEAWSVEKT FDHMKQHAAIGAQILA.N.S......SSPLLQLAHKLAIEHHEKWDGSGYPNGLKGEQISVEGRIVAIADVFDALTSKRPYKEAWGVEEA FAHMKEHAAIGAKILA.N.S......PSPLLQLAHVLAIEHHEKWDGTGYPNGLKGEEISIEGRIVAIADVFDALTSKRPYKEPWSIEQT FTHMKEHALIGAQILA.N.S......SSPLLQLAHVLAIEHHEKWDGTGYPNGLKGEAISLEGRIVAIADVFDALTSKRPYKEPWSIEQT FAHMKEHAQIGAKILA.N.S......TSPLLQLAHLLAMEHHEKWDGTGYPNGLKAEQISIEGRIVAIADVFDALTSKRPYKQAWSVDEA FEHMKEHALIGAKILA.N.S......SSPLLQLAHTLAIEHHEKWDGTGYPYGLKGDEISIEGRIVAVADVFDALTSKRPYKDAWSVEKA FEHMKQHAVIGAKILE.N.S......SSPLLQLAHKLALEHHEKWDGTGYPLGLKGEEISIEGRIVSIADVFDALTSVRPYKDAWPVEKA FQLMKTHAQIGAHILQ.G.S......SSPLIQLAHTLALEHHEKWDGSGYPQGLKGEEISLEARIVSIADVFDALTSKRPYKQAWPIDDA YRQMQTHASIGADILA.G.S......SSALIQLAHRLALEHHERFDGSGYPNGLQGEEISIEGRICAIADVFDALTSKRPYKEPWPIDKA FEEMKKHPQIGAEILG.E.S......DSDLIELAKVVSLTHHEKWDGSGYPKGLAGEEIPIHGRIVALADVFDALTSKRPYKEAWSIDKT FAIMKKHPEIGVEILG.D.D......DSELIALAKTVALTHHEKWDGSGYPKGLKGEDIPIEGRIVALADVFDALTSKRPYKEAWSIEKT FEVMKTHPQIGADIIG.E.D......DSLLMQMARTVALTHHEKWDGSGYPRGIAGEAIPVEGRIVAIADVFDALTSDRPYKKAWSIEDT MRIMQQHPLIGAEILA.N.T......NSELIKLAHSVALHHHEKWDGTGYPAGIKGEAIPIEGRIVAVADVFDALTNKRPYKEAWPVDKT WEVMQTHVQIGADIIN.N.G......DSLLLQMAQEIALYHHEKWDGNGYPHGLKGEEIPLSARIVAIADVFDALTSERPYKKAWPTEKA WAVMQTHVKIGADIIA.D.S......DTRLMQMAQEIALYHHEKWDGSGYPHGLKGEDIPLSARIVAIADVFDALTSERPYKQAWPIEKA WEIMKTHVDIGADLLA.G.T......DVPLLSMARNIALTHHEKWDGSGYPRGLKAEEIPIEGRICAICDVFDALTSERPYKRAWPIEDA WTTMQKHVEFGVEILGRQ.G......DSKLMRMAIQVAQYHHEKWDGSGYPKQIAGEDIPLVGRIAAVADVFDALTAERPYKKAWSVDEA WTIMQKHVEFGVEILGRQ.S......DSKLMQMAIQVAQYHHEKWDGSGYPNQIAGEDIPLVGRIAAVADVFDALTAERPYKKAWSVDEA WTIMQKHVEYGVEILGRQ.S......DSKLMRMATQVAQYHHEKWDGSGYPNQIAGEAIPLVGRIAAVADVFDALTAERPYKKAWSIEEA YEIMKTHTTIGYNLLR.G.S......NRELLRTAAHIAHEHHEKWDGTGYPRGLKGEEIHLYGRITAVADVFDALGSDRVYKKAWELERI FEIMKSHASIGYNLLK.N.S......KRHILKAASIIANEHHEKWNGRGYPNGKKGEEIHIYGRITAIADVFDALASDRCYKKAWELDRI WVIMKSHTEIGHRMLS.H.S......ERPILKTAAVVAYEHHEKWNGKGYPRGLKGDDIHIFGRITAIADVFDALGSDRVYKKAWDLDRI REIMNTHAALGYEMLK.H.S......NRPLLKMAAIVANEHHEKWDGSGYPKGLSGENINIYGRITALADVFDALGSDRVYKKAWDDERI WVIVKMHPKRGYEMLK.Q.S......SLAVMNISAIIALTHHEKWDGTGYPEGLKGDDIHIYGRITAIADVFDALGSDRCYKAAWPLDKI FELMKNHTTFGWEIFN.K.S......KHQLLQAAALISYQHHEKWNGTGYPRGISGEDIHVFGRITAIADVFDALCHDRIYKKAWSVEDT YLEMQRHVEIGGEILEGH.D......SN.VLKTAYEIALTHHEKFDGKGYPKGLSGKDIPISGRIVAIADVFDALTSERPYKEAWPVEKA FTIMKQHAMYGWEILK.D.S......SSQLVRLAALIARTHHEKFDGSGYPEGLAGDKIPLEGRIVAVADVFDALTSTRPYKTPWPVDKA FEVMQHHAAIGYEILS.H.S......ASRVLQLGAEIALSHHEKYDGSGYPKRLHGEDIPLFGRIVAVADVFDALTSERPYKKAWPLDTA WEVMKSHAVIGGNILS.M.G......SSEYINMGSVIALSHHEKWDGSGYPSGLSGEDIPLPGRICAVADVFDALTSKRPYKEPFSVEKS 95
PA4781
170
LQQLQDAVIEALATLGDLRDNPRSRHLPRIERYVRLLAEHLAAQ.RAFADELT.PEAVDLLSKSALLHDIGKVAVPDRVLLNPGQLDAAD LQQLQDVTIEALATLGDLRDNPRSRHLPRIERYVRLLAERLATQ.AAFADALT.PEAVGLLSKSALLHDIGKVAVPDRVLLNPGQLDAAD LQAIQDATLEAMATLCDLRDNPHSQHLVRIEHYMRLLCCTLALR.NEFSGELS.VENIELLARSAQLHDIGKVAVPDRILHSPGQLDEAD LQAIQDATLEAMATLCDLRDNPHSNHLVRIEHYMRLLGSSLSLH.PEFAAELS.VENIELLVRSAQLHDIGKVAVPDRILHNPGQLEEAD LQAAQDATLEAMATLCDLRDNPHSRHLLRIEQYMRLLADALARR.EGEAGGLD.LEQIELLARSAQLHDIGKVAVPDRVLLNPGQLSPED IQHLHDATIEALAGLADMRDNPDGNHLVRIELYMRLLGTALARQQPGMAEELT.EERINLMAKSALLHDIGKVALPDRVLLNPGQLEGDD ...LQDVSMMAMALLSESRDYETGAHLQRTSRYVRALAINLYAK.AIFAKELT.LDSIFLLSKSALLHDIGKLTIPETVLGKRGKLTSAE ...VQDVSIHALAHLAEIRDPETGNHLRRTQGYVRALAEYLSDH.PRFADFLT.PHAIDLLVKSAPLHDIGKVGIPDHILLKPGKLTPEE ....QNVAIWALASLAETRDNETGNHIKRTQSYVKLLAEQLSGH.PRFDQYLT.ERNIDMICKSAPLHDIGKVGIPDHILLKAGKLTDDE ...IQDVTIMAMASLAETRDNETGNHLRRTQHYVRRLAQALRHH.PRFCDELN.DETIELLFKSAPLHDIGKVGIPDRILLKPGKLTAEE VSMIQDVTIMAMASLAETRDNETGNHIRRTQNYVRILARQLQGH.PRFAAFLS.DANIELLYKSAPLHDIGKVGIPDRILLKPGKLTPEE LAELQDATIRAMASLAETRDNETGNHIRRTQHYVEALARKLQDH.PRFRDELT.EDAIQVIFKSAPLHDIGKVGIPDRILLKPGKLTVEE MAELQDATIRAMASLAETRDNETGNHIRRTQHYMEALARHLQNH.PRFKDELT.DAAIETIFKSAPLHDIGKVGIPDRILLKPGKLTPEE IEAIQDVTILAMASMAETRDNETGNHIRRTQFYVRALADKLKKH.PRFSSFLT.ERCINMLFKSAPLHDIGKVGIPDHILLKPGRFTPEE LAAIQDVAILAMASLAETRDNDTGNHIRRTQFYVKALAIHLQPH.PRFRDFLD.DNTIDLLFKSAPLHDIGKVGIPDRILLKPGRFDAAE ...LQDVTIHTMASLAETRDSETGNHIRRTAHYVKTLAEKLRTN.PRFSDFLT.DKNIELLFKSAPLHDIGKVGIPDRILLKPGRFEGNE ITALQDVTIHAMASLAETRDNETGNHIRRTQYYVKTLAEKLRFH.PRFAHFLNDDETIELLFKSAPLHDIGKVGIPDRILLKPGRFEPEE VQVIQDVTIMALASLAETRDHETGNHLRRTQNYVRALALELRNH.PNYRPQLS.DETVQLLYKSAPLHDIGKVGIPDHILLKPGPLTPEE LEALQDATIIAMASLAETRDNETGNHIRRTQRYIEVLANQLLLE.GQYADQLT.PESIKALYKSSPLHDIGKVGIPDHVLLKPGKLTEEE LEALQDATIIAMASLAETRDNETGNHIRRTQRYIKVLAEQLRLD.GQYADQLT.PESILALYKSSPLHDIGKVGIPDHVLLKPGKLTEEE LEMLQDAIIVAMASLAETRDNETGNHIRRTQHYVRALANQLVSE.GRYMDQLT.PAVIELLFKSAPLHDVGKVGIPDQVLLKPGKLTEDE LELLQDATILAMASLAETRDNDTGNHIRRTQKYVKILAETMAES.PKYASKLD.SETIEALYKSAPLHDIGKVGIPDNILLKPGKLTDEE MSQLQDVTIQAMASLAETRDQETGNHIRRTQLYVKLLATILSGK.EKYKQILT.PEVINIYYKSAPLHDIGKVGIPDNVLLKPARLTAEE LEQTQDITIRALASLAETRDNETGNHIKRTQYYVRALAEHLRHH.PDFSAELD.DTAIDLLFKSAPLHDIGKVGIPDAILLKPGRLDTEE LQQTRDIAIHSLATLAETRDNETGAHILRTQYYVKALAEALQNE.QGFSNLLS.PQIIDLIYKSAPLHDIGKVGIPDAILLKPGQLDDEQ LKKAHEDVIYRLSHATKFKDPETQNHIIRVGLYCEILAREAG.....LDEEDV.....ELVKLAAPMHDIGKVGIPDRVLLKPGKLNDEE IKDAYREAVMRLSHAAEYKDPETYNHIVRVGLFARLMAERLG.....LEKEVR.....DSIMLAAPMHDIGKIGIPDAILLKKGKLNDWE LNDAYKETVLKLSHAAEYKDKETKNHILRTGLYAKVVGRELG.....FDDDEI.....NILFLATQMHDIGKIGIPDRILLKPGKLTEEE LKQAHVDLVERLGRAAEYKDTDTGEHIVRMSQYSKVLALALG.....MDEQRA.....ELLRQAAPMHDVGKIGIPDAILLKPGKLTSEE LKQAHIDLVQRLGRAAEYKDTDTGEHIVRMSQYSKVLALALG.....MPEHQA.....ELLRQAAPMHDVGKIGIPDAILLKPGRLTSEE LKQAHVDLVHRLGRAAEYKDTDTGEHIARMSQYSKLLALEFG.....MGEQQA.....ELLRQAAPMHDVGKIGIPDAILLKPGRLTPEE LKQAHVDLVERLGRAAEYKDTDTGEHIARMSQYSKVLALALG.....MNEQHA.....ELLRQAAPMHDVGKIGIPDSILLKPGKLTPDE LKQAHVDLVQRLGRAAEYKDTDTGEHIIRMSQYSKVLALALG.....MDEQHA.....ELLRQAAPMHDVGKIGIPDAILLKPGKLTADE LKQAHVDLVHRLGCAAEYKDTDTGEHIIRMSKYSKILALAYG.....MDEQHA.....ELLKQAAPMHDIGKIGIPDAILLKPGKLTDEE LKQAHIDLVQRLGRAAEYKDTDTGEHIVRMSQYSKVLALGIG.....MSENQA.....ELLHQAAPMHDVGKIGIPDAILLKPGKLSEQE LQLAHIDLINRLGRAAEYKDTDTGEHIARMSRYSKVLALAYG.....MTEYQA.....EQLKQAAPMHDVGKIGIPDSVLLKPGRLDANE LKQAHIDLVQRLGNAAEYKDEDTGEHILRMSHYSKLLALEAG.....ISEEHA.....ELIRQAAPMHDIGKIGIPDAILLKPERLSEDE LKAAQLELIQRLSHAAEYKDNETGQHIIRMSRYCHIIARAYG.....FNEAHA.....ESLLLAAPMHDIGKIGIPDNILLKPGRLDQDE LRRTRLQVIQRLGRASEYKDNETGMHVVRMSHYSRLLALACG.....FSENAA.....DDIFNAAPMHDIGKIGIPDNIMLKPARLTEEE LRETRLQIIQRLGRASEFKDNETGMHVMRMSHFAKIIALAYG.....FSEQRA.....DLLLHTAPMHDLGKIGIPDHIMLKPGKLSEEE LKVTRLKIIQRLGRAAEFKDNETGMHVMRMSHYAKVLARAYG.....LGESHA.....DMLLHAAPMHDIGKIGIADHILLKPGKLTEEE LKKTHLQLIQRLGRAAEYKDNETGMHVMRMSHISKILALALG.....FNEDFA.....DKLLQAAPMHDIGKIGIPDHILLKPGRLDDEE LESSRLEVIQRLGRAAEYKDNETGMHVIRMSHYSKILAQQLD.....VSERWI.....ELVFQASPMHDIGKIGIPDAVLRKPGKLDKDE LEKTRLEVIQRLGRAAEYKDNETGMHVIRMSHYSRILAQQLD.....VSERWV.....ELVFQASPMHDIGKIGIPDAVLRKPGKLDAEE LEQSRLEIIARLGRAAEYKDNETGNHILRMSHFAGLLAKAAN.....LGEKFS.....ASIELAAPMHDIGKIGIPDYILLKPGKLDDDE ...........LGRAAEFKDNETGMHVKRMSHYCEVLAKALG.....MTDEDA.....ETLRDAAPMHDIGKIGIPDSVLLKPGKLDADE ...........LGRAAEFKDNETGMHVMRMSHYCEILAKALG.....MTDEDA.....ETLRDAAPMHDIGKIGIPDSVLLKPGKLDADE ...........LGRAAEFKDNETGMHVMRMSHYCEILAKALG.....MTDEDA.....ETLRDAAPMHDIGKIGIPDSVLLKPGKLDADE ...TQKEIIFRMGEIGESRSKETGNHVKRVAEYPRILALGLG.....LPQEEA.....EKIRVASPMHDIGKVAIPDAVLKKPGKLTDEE IEETQKEIIYTMGEIGESRSKETGNHVKRVAEYSRILADGLG.....MKREET.....ELIKMASPMHDIGKVAIPDDILKKPGKLTDEE IEKTQKEIIFTMAEAGEMRSKETGNHVKRVAEYSKLLALGYG.....LSEEEA.....EMLRMASPMHDIGKIAIPDSVLLKPGKLDADE IENTQKEVVFTMGAIGESRSKETGNHVKRVAEYSKLLALYYG.....LDEKEA.....ELLKQASPMHDIGKVAIPDAILNKPGRFDENE ITESQKEMVYRLGEVVESRSNETGNHVKRMAHYSELLALLVG.....LDKTEA.....ELIKTASPMHDIGKIAIPDAILTKPGKLTPEE ...TQEEVLQTLGELGEWRSKETGDHVNRVSLFSELLARAHG.....CDEENV.....ALLKMASPMHDIGKVIIPDAILLKPGKLSDEE LEDAHHEIIYRLGRASEYRDNETGNHVKRVSYFSELIALEAG.....LDKKEA.....HMIRIASPMHDVGKIGISDTIMMKPGKLTDEE ..RAAEELVMRLSKAAEYRDPETGAHIERMARYSALIARRMG.....LPNEAV.....ERLELAAPMHDVGKVGIPDMILLKPDRLSETE ......ETVFRLSKAAEYRDPETGGHILRMAHYSELIARELG.....CSTQEQ.....EVLRDAAPMHDIGKVGIADHILLKPGRLTPEE LDEAHVETIHHLCAAAEYKDEETADHLVRMAEYSRILAEKSG.....LDVDTV.....QLIHTSSPMHDIGKIGIPDSILLKPGKLTAEE
100
105
110
115
120
125
130
135
140
145
150
155
160
165
170
175
180
15599975_P.aeruginosa_PAO1 340
15599975_P.aeruginosa_PAO1 152988376_P.aeruginosa_PA7 472324017_P.denitrificans_ATCC 516091093_P._nitroreducens 575528596_Pseudomonas_sp._M1 544802557_P.alcaligenes 517531475_A.acidaminophila 288940945_A.vinosum_DSM_180 34496338_C.violaceum_ATCC_1247 493140715_C.basilensis 372487460_D.suillum_PS 498144707_A.radicis 407939109_Acidovorax_sp. 71909165_D.aromatica_RCB 517819128_A.toluclasticus 522196548_O.bacterium_AB_14 515524292_C.agarivorans 34497952_C.violaceum_ATCC_1247 491656777_V.mimicus 496733920_Vibrio_sp._RC586 491493257_A.diversa 549689089_V.nigripulchritudo 498066214_P.rubra 518476831_N.itersonii 511824815_A.albus 225850538_P.marina_EX-H1 495562704_Hydrogenivirga_sp. 551219240_Desulfurobacterium_s 489051231_P.haloplanktis 495447713_Pseudoalteromonas_sp 567931853_P._sp._NW_4327 495388329_Pseudoalteromonas_sp 77360093_P.haloplanktis 515078141_P.haloplanktis 493845294_A.bacterium_TW-7 584421140_P.lipolytica 519054894_G.chinensis 551324596_P.ruthenica 496119206_G.lipolytica 498247266_P.spongiae 119776243_S.amazonensis_SB2B 519026623_Methylotenera_sp._1P 490545238_V.orientalis 490886322_V.tubiashii 497412599_M.lonarensis 515633726_V.crassostreae 516227747_Vibrio_sp._624788 515642306_V.splendidus
350
360
VLLIQAGAGSEFDPRLVEAFVAVADAFAEVARRYADSA VLLIQAGAGSEFDPRLVKAFVAVADAFAEVARRYADSA AARIKAGAGSQFDPRMVEAFSEAAEGFASIALRYADS. AERIKAGAGSQFDPRVVEAFREVAEGFANIAQRYADS. RQQILAASGSHFDPQVVEAFAEVADGFAAIAIRYADDA VMRISAGSGSEFDPLVVMAFLEVADGFALIAQRHADS. VRIIKAAAGTQFDPVIVEAFLETSDSFAAIAMQFPD.. YTIIVAGRGTHFDPDVVDAFIIRFDEFQDIAIRYGEA. KDVIVAGRGTHFDPDVVDAFLAVEQAFVEVALAYADA. VRMIGEGRGGHFDPDMVDAFLAIAPEFLAISQRYADTA VQIISEGRGKHFDPDMVDAFLQVAEDFRSIAATFADT. VEIIRDGRGSHFDPDMVDAFLALSEEFRRIAQRFAD.. VEIIRDGRGSHFDPDMVDAFLTLSEEFRRIAQQFADA. VQMIIDGRGSHFDPDMVDAFVELQDEFLAISQRYADS. AKIIREGRGSHFDPDIVDAFIELQDEFRVIAARFSD.. VAIIIEGRGQHFDPDMVDAFLELQQDFIDIARRYADS. VQIIIDGKGSHFDPDMVDAFIDLADEFKNIAARYADS. VGIIRDDSGRHFDPAVVKAFLQVAEQFREIARLYSDA. LAIIEEGRGQHFDPIIVDAFLSVEDKFKQIAKELAD.. VTIIEEGRGHHFDPAIVAAFLKIERQFRQIAQELSD.. VSIIREGRGQHFDPVVVDAFLAIEGHFQEIARRFHD.. VEIIESSSGSHFDPDVVEAFRRVEQTFDEIAHRFAD.. IAIMKEGRGSHFDPELLDEFLAQESAFFKISQQYRD.. VAMIREGSGTHFDPAIIDAFLTLQEQFREIAQRYAD.. KQIILEGKGSHFDPAVVEAFCRCEQQFVEIAAQYSD.. VRFFKEQKGKHFDPFLTDIFLKNIDQMFSIKRELR... VEHMNSMKGKQFDPQLIELFFSNLEEVVDIR....... IKLLEEEAGTHFDPRIAEIVIRKADELFEIKESL.... VLHMQEQAGTHFDPALIELLVKNLDDIIAIKN...... ILHMQDQAGAHFDPALIELFVSKLDDILAIKN...... LEHMQAQAGKHFDPHLINLFVNKLDAIIAIKN...... LEHMQQQAGKHFDPQLVDLFISKIDTILAIKK...... LEHMQQQAGKHFDPQLVELFIGKIDDIIAIKN...... ISHMRTQAGKHFDPQLIALFEGQLDAILEV........ VAHMKEQAGSHFDPAIIDLLVNKLDEILNIKQ...... VELLQAESGKHFDPQLVDLFIGQIDAIIEIKN...... FKLIEDQAGLHFDPELVKLFLQLKNEILAIKKQ..... VQLLQEEKGKHFDPELVDIFMACLDDILAVKAQ..... MEYLQQQKGKHFDPQLVDLFQQNLDKMLEIK....... MAFLHEQKGKHFEPKLVDLFSQELDKILAIK....... LRLMREQKGRHFEPLLVELLEQNLDKILDIKQ...... MDFFKEQRGKHFDPSIVDALVRELDHILQ......... IALLEEEAGSHFDPTLVPIFIAQLDQLLEIKESFK... TALLEEEAGSHFDPTLVPIFLQQLDEILKVKESFK... LQYLRQQAGSHFDPDLIPLFEKILDEVLTFRAQ..... LSLFEEQKGKHFDPTIVDMFFLKLPEILAIKEKFR... LSLFEDQKGKHFDPKIVELLFENLPQILAIKEKFK... LSLITEQKGKHFDPTIVDLFLVNLPEILAIKEKFK...
Figure S3. Multiple sequence alignment. Non-redundant (identity threshold < 95%) multiple sequence alignment of 58 HD-GYPs sequences closely related to PA4781.
3
FIGURE S4
Figure S4. Determination of the active site metal. On the left, X-ray fluorescence spectra of non-diffracting PA4781 crystals. The excitation wavelength was 11.3 KeV (1.100 Å). The inset panel shows the region of interest, where emission due to Nickel was observed only for the sample (red) and not in the controls (green and blue). On the right, results of the ICP-MS analysis performed on “as purified” PA4781 used for crystallization trials, after denaturation of the protein.
4
FIGURE S5
Figure S5. Plot of ΔG, ΔH, and −TΔS derived from the ITC data obtained from metal binding to L-site of semi-Apo PA4781. The thermodynamic signature indicates that binding of metal to L-site is enthalpy-driven, accounting for polar interactions which take place, while the unfavourable entropic factor suggests a conformational change occurring upon binding. In such a context, binding of Cu2+ represents an exception, since the interaction is both enthalpically and entropically favoured, being the negative entropic factor indicative of a more hydrophobic character of this interaction. The Zn2+ experiment is not included given that a displacement equilibrium takes place aside from binding to Lside.
5
FIGURE S6
Figure S6. Titration of “as purified” PA4781 with Zn2+. Analysis was performed by titrating 5 μM PA4781 with a 100 µM Zn2+ solution (p) in 150 mM NaCl, 20 mM TRIS pH 7.2. A) Graph shows the integrated energy values normalized for injected protein. Binding isotherms were fitted using a single binding site model (n=1.08), yielding a KD=0.17 µM. The measurement was done in duplicate, error is within 5%. As control, the “as purified” PA4781 was titrated Ni2+ r; as expected no binding was observed. B) Thermodynamic profile obtained from fitting the data reported in A). According to the hypothesis of the displacement equilibrium (Fig. 5), the entropic factor is minimal as compared to data reported in Figure S5. Given that the affinity for both Ni2+ and Zn2+ is comparable, as assayed with the fully-Apo protein, displacement of Ni2+ by Zn2+ is most likely kinetically favoured, assuming that the Ni2+ binding is characterized by rapid dissociation constant (as compared to Zn2+). Nevertheless, the presence of a third M-site, yet to be identified, which allosterically controls metal binding to the active site (according to the sequential binding model of binding proposed for ITC data reported in Figure 4), cannot be ruled out, even though it could be strongly selective for Zn2+ and located beyond the portion of protein found in the crystals.
6
FIGURE S7
Figure S7. PDE activity of PA4781 in the presence of different metals. Chromatograms showing the nucleotide content of the reaction of PA4781 with c-di-GMP (Panel A) or pGpG (Panel B). More in detail, in A) pGpG and GMP formation obtained after incubation of 5 μM phosphorylated PA4781 with 120 μM c-di-GMP is reported. In B) GMP formation obtained after incubation of 5 μM phosphorylated PA4781 with 80 μM pGpG. The nucleotide content was evaluated on a C8 RP-HPLC after 20 min upon substrate addition, as previously published [15]. As control, the reaction of the “as purified” protein (holo in the figure), handled as reported in [15], is also shown. In agreement with [15], the PDE activity of the “as purified” enzyme is very little. Nevertheless, reconstitution of PA4781 with different metals does not overturn this result and fails to trigger an effective catalysis. In light of these results, it is likely that the nature of the metal present in the active site does not affect significantly the affinity for the two nucleotides. In order to prove this statement, attempts to measure the dissociation constant of PA4781 with pGpG and/or cdi-GMP were done, but unsuccessfully. Indeed, experimental limitations did not allow us to obtain this values: i) titration of phosphorylated PA4781 under non-cycling conditions (i.e. in the presence of CaCl2) with c-di-GMP or pGpG could not be carried out, due to precipitation of calcium phosphate; ii) binding studies on the truncated version of the protein PA4781-G112start (lacking the REC domain), could not be carried out given that this construct was purified as a nucleotide-bound form. All the attempts aimed at removing the nucleotide portion (such as dialysis and gel filtration) were unsuccessful.
7
FIGURE S8 A
B
PA4781 fully Apo PA4781 + Zn PA4781 + Ni PA4781 + Mn PA4781 + Co PA4781 As-Purified (Ni) 4781-G112start
PA4781 fully-Apo (CD) PA4781 + Zn (CD) PA4781-G112start (CD) 0
-1
-1
400
380
-0.5
2
2
-0.5
4
-1
-1.5
transition midpoint -2
360 -1 protein precipitation
340
-1.5 320
High Tension voltage (V)
4
Mean Residue Ellipticity (10 deg cm dmol )
0
Mean Residue ellipticity (10 deg cm dmol )
PA4781-fully-APO (HT) PA4781 + Zn (HT) PA4781-G112start (HT)
protein precipitation
-2
300
Apparent Tm -2.5
-2.5 290
300
310
320
330
340
350
290
360
T (K)
300
310
320
330
340
350
280 360
T (K)
Figure S8. Thermal denaturation of PA4781 bound to different metals. A) Thermal denaturation experiments qualitatively showing the effect of metal binding on protein stability. It is evident how Co2+ shifts the apparent Tm (see supplementary methods) of about 5 ˚C, while Mn2+, Zn2+ and Ni2+ of 12 ˚C. Notice that, at the end-point, the full length proteins retain more than 50% of the dichroic signal, while the mutant PA4781-G112start (lacking the REC domain and bound to Ni2+) is 100% unfolded. However, the amplitude of the transition (Δ[θ]) is identical in both cases, indicating that while the HD-GYP domain undergoes thermal denaturation, the REC domain retains most of its structure at 85 ˚C. Notice that, during the heating process, PA4781 fully-Apo, PA4781 + Zn2+ and PA4781-G112start precipitated as illustrated in panel B) that shows both the dichroic signal (continuous line) and the absorbance (dashed line) of the sample as a function of T. The arrows indicate the approximate T at which the samples started to precipitate. While for PA4781 fully-Apo and PA4781 + Zn2+, precipitations occurs at high T (> 80 ˚C), PA4781-G112start precipitates during the unfolding process. These results confirm the instability of the truncated mutant over the full length and the peculiar behaviour of zinc over the other metals.
8
FIGURE S9
Figure S9. Scheme of the allosteric regulation of PA4781. On the Left: Hypothetic allosteric regulation scheme for PA4781 activation: following phosphorylation of the CheYLike regulatory domain (REC), the signal is allosterically transmitted to the HD-GYP domain by a torsion of the S-helices (in green), which induces a conformational change of the helices-bundle at the dimeric interface (comprising also α2 and α3), ultimately affecting the Lid region. On the Right: top-view of the helices-bundle at the dimeric interface, and a section of the protein surface (coloured by electrostatic potential), showing how the Lid-region limits the access to the active site cavity; small conformational changes in this region may result in large changes in substrate affinity.
9
Supplementary Methods Circular dichroism and thermal denaturation: CD spectra were measured for 5 µM (semi-Apo PA4781) and 10 µM (fully-Apo PA4781) protein samples in 150 mM NaCl and 20 mM Tris/HCl buffer pH 7.6, using a Jasco CD spectrometer at 20°C using a 1 mm quartz cuvette (Hellma). Holo-samples samples were obtained by incubating with a 4-fold excess of divalent metal ions (20 µM and 40 µM, respectively) for 30 minutes at RT. Thermal denaturation profiles were acquired by heating protein samples (10 µM) from 15 to 85°C, with a heating rate of 1 deg/min controlled by a Jasco programmable Peltier element, monitoring the dichroic activity at 220 nm every 1 °C. Given that PA4781 is a dimeric multi-domain protein, data were not fitted to a simple two state equation (i.e. implying only a folded and an unfolded state), and reported values are only a graphical extrapolation of the T corresponding to the midpoint of the observed transitions (apparent Tm).
ICP-MS Chemicals: nitric acid for trace analysis ≥ 67 % (Promochem, LGC Standards GmbH, Wesel, Germany) and deionized water produced by a deionizator Elga Lab Water Purelab Plus were used. All dilutions were obtained using 1% nitric acid for trace analysis as diluent. A multi-element standard solution of
65
Cu,
55
Mn and
60
Ni at the concentration of
1 - 2 - 5 µg/L was prepared by dilution from a multi-element stock solution at 1.000 ± 0.005 µg/mL for each metal, purchased from Ultra Scientific Italia (Bologna, Italy); a multielement standard solution of
57
Fe and
66
Zn at the concentration of 10 - 20 – 50 µg/L was
prepared by dilution from a multi-element stock solution at 10.000 ± 0.05 µg/mL for each metal, purchased from Ultra Scientific Italia (Bologna, Italy) and standard solution of
59
Co
at the concentration of 5 - 10 - 25 µg/L was prepared by dilution from a multi-element stock solution at 5.01 ± 0.03 µg/mL, purchased from SCP Science (Courtaboeuf, France). Standard solution of
89
Y as internal standard at the concentration of 10 µg/L was prepared
by dilution from a stock solution at 1,000 ± 0.002 µg/mL, purchased from Panreac Química (Barcelona, Spain).
ICP-MS Method of analysis: an internal standard method was adopted, using Y as the internal standard and the multi-standard solutions above described. Whole procedure
10
blank tests were performed, in order to assess the absence of any contamination occurring from reagents and materials.
11
