Study of Adenylyl Cyclase-GαS interactions and identification of novel AC ligands

AppalarajuJaggupillia,b, PremnathDhanaraja,b, Alexander Pritcharda,c, John L. Sorensena,c, ShyamalaDakshinamurtia,d,e and PrashenChelikania,b,e*

aManitoba Chemosensory Biology (MCSB) Research Group, bDepartment of Oral Biology,University of Manitoba, Winnipeg, Canada, MB, R3E 0W4, cDepartment of Chemistry, University of Manitoba, Winnipeg, MB R3T 2N2, Canada, dDepartments of Pediatrics, Physiology, University of Manitoba, eBiology of Breathing, Children’s Hospital Research Institute of Manitoba (CHRIM)

SUPPLEMENTARY INFORMATION

* Correspondent Author: Dr. PrashenChelikani, D319, Department of Oral Biology, and Manitoba Chemosensory Biology (MCSB) Research Group, University of Manitoba, Winnipeg, MB R3E 0W4. Canada 204-789-3539 (phone), 204-789-3913 (fax), email:

Supplementary figure 1. Multiple sequence alignment of human ADCY sequences and PDB:1CJK using CLUSTAL Omega. Conserved residues for forskolin binding are highlighted in red and predicted nitrosylatable cysteine in yellow.

ADCY9 ------

ADCY3 ------

ADCY8 ------

ADCY1 ------

ADCY4 ------

ADCY7 ------

ADCY6 ------

ADCY2 ------

1CJK ------

ADCY5 MSGSKSVSPPGYAAQKTAAPAPRGGPEHRSAWGEADSRANGYPHAPGGSARGSTKKPGGA

ADCY9 ------

ADCY3 ------

ADCY8 -----MELSDVRCLTGSEELYTIHPTPPAGDGRSASRPQRLLWQTAVRHITEQRFIHGHR

ADCY1 ------

ADCY4 ------

ADCY7 ------

ADCY6 ------MSWFSGLL------VPKVDERKTAWGERNGQKRSR------

ADCY2 ------MWQEA------

1CJK ------

ADCY5 VTPQQQQRLASRWRSDDDDDPPLSGDDPLAGGFGFSFRSKSAWQERGGDDCGRGSRRQ--

ADCY9 ------MASPPHQQLLHHHSTEVSCD------SSGDSNSVRVKINP

ADCY3 ------MPRN

ADCY8 GGSGSGSGGSGK------ASDPAGGGPNHHAPQLSGDSALPLYSLGPGERAHSTCGT

ADCY1 ------

ADCY4 ------

ADCY7 ------

ADCY6 -RRGTRAGGFCTPRYMSCLRDAEPPSPTPAGPPRCPWQDDAFIRRGGPGKGKELGLRAVA

ADCY2 ------

1CJK ------

ADCY5 -RRGAASGGSTR------APPAGGGGGSAAA------AASAGGTEVRPRSVE

ADCY9 KQLSSNSH--PKHCKYSISSSCSSSGDSGGVPRRVGGGGRLRRQKKLPQLFERASSRWWD

ADCY3 QGFSEPEYSAEYSAEYSVSLPSD------PDRGVGR------THEISVRNSGSCL-

ADCY8 KVFPERSG------SGSASGSGGGGDLGFLHLDCAPS------NSDFFLNGGYSYR

ADCY1 ------MAGAPRG-GGGGGGGAGEPGGAER------AAGTSRRRG-----

ADCY4 ------

ADCY7 ------MP------

ADCY6 LGFEDT------EVTTTAG------GTAEV------APDAVPRSGRSCWR

ADCY2 --MRRR----RYLRD----RSEEAAGGGDGLP------

1CJK ------

ADCY5 VGLEERRGKGRAADELEAGAVEGGEGSGDGGSSADSGSGA------GPGAVLSL-GACCL

ADCY9 ---PKFDSVNLEEACLERCFPQTQRRFRYALFYIGFACLLWSIYFAVHMRSRLIVMVAPA

ADCY3 -CLPRFMRLTFVPESLEN------LYQTYFKRQRHETLLVLVVFA

ADCY8 GVIFPTLRNSFKSRDLER------LYQRYFLGQRRKSEVVMNVLD

ADCY1 ---LRACDEEFACPELEA------LFRGYTLRLEQAATLKALAVL

ADCY4 -MARLFS--PRPPPSEDL------FYETYYSLSQQYPLLLLLLGI

ADCY7 AKGRYFLNEGEEGPDQDA------LYEKYQLTSQHGPLLLTLLLV

ADCY6 RLVQVFQSKQFRSAKLER------LYQRYFFQMNQSSLTLLM-AV

ADCY2 ------RSRDW------LYESYYCMSQQHPLIVFLLLI

1CJK ------

ADCY5 ALLQIFRSKKFPSDKLER------LYQRYFFRLNQSSLTMLM-AV

ADCY9 -L---CF------LLVCVGFFLFTFTKL------YA

ADCY3 ALFD-CYVVVMCAVVFSSDK------LASL--A--VAGIGLVLDIILFVLCKKGLLP

ADCY8 -VLT-KLTLLV--LHLSLASAPMDPLKG---I------LLGFFTG---IEVVICALVVVR

ADCY1 SLLAGALALAE--LLGAPGPAPGLAKGSHP-----VHC------VLFLALLVVTNVRSLQ

ADCY4 VLCA-LAALLA--VAWASG----RELTSDPSFLTTVLCALGGFS----LLLGLASREQRL

ADCY7 AATA-CVALII--IAFSQG----DPSRHQA-ILGMAFLVLAVFAALSVLMYVECL----L

ADCY6 LVLL-TAVLLA--FHAAPA----RPQ---PAYVALLACAAA----LFVGLMVVCNRHSFR

ADCY2 VMGS-CLALLA--VFFALG----LEVEDHVAFLITVPTALAI---FFAIFILVCIESVFK

1CJK ------

ADCY5 LVLV-CLVMLA--FHAAR------PPLQLPYLAVLAAAVGV---I-LIMAVLCNRAAFH

ADCY9 ----R---HYAWTSLALTLLVFALTLAAQFQVLTPVSGRGDSSNLTATARPTDTCLSQVG

ADCY3 DR------VTRR-VLPYVLWLLITAQIFSYLGLN------FARA-HAASDT--

ADCY8 ----KDTTSHTYLQ-YSGVVTWVAMTTQIL------AAGLGY-GLLGDG--

ADCY1 VPQLQQVGQLALLF-SLTFALLCCPFALGGPARGSA------GAAGGP-ATAEQG--

ADCY4 ----Q-----RWTR-PLSGLVWVALLALGH--AFLF------TGGV-VSAWDQ--

ADCY7 ----R-----RWLR-ALALLTWACLVALGY--VLVF------DAWTKA-ACAWEQ--

ADCY6 ----Q-----DSMW-VVSYVVLGILAAVQV--GGAL------AADP-RSPSAG--

ADCY2 ----K-----LL-R-LFSLVIWICLVAMGY--LFMC------FGGT-VSPWDQ--

1CJK ------

ADCY5 ----Q-----DHMG-LACYALIAVVLAVQV--VGLL------LPQP-RSASEG--

ADCY9 SFSMCIEVLFLLYTVMHLPLYLSLCLGVAYSVLFETFGYHFRDEACFPSPGAGALHWELL

ADCY3 -VGWQVFFVFSFFITLPLSLSPIVIISVVSCVVHTLVLGVTVAQQQQEELKGMQLLREIL

ADCY8 -IGYVLFTLFATYSMLPLPLTWAILAGLGTSLLQVILQV----VIPR---LAVISINQVV

ADCY1 -VWQLLLVTFVSYALLPVRSLLAIGFGLVVAASHLLVTATL---VPA---KRPRLWRTLG

ADCY4 -VSYFLFVIFTAYAMLPLGMRDAAVAGLASSLSHLLVLGLYLGPQPD---SRPALLPQLA

ADCY7 -VPFFLFIVFVVYTLLPFSMRGAVAVGAVSTASHLLVLGSLMGGFTT---PSVRVGLQLL

ADCY6 -LWCPVFFVYIAYTLLPIRMRAAVLSGLGLSTLHLILAW----QLNR---GDAFLWKQLG

ADCY2 -VSFFLFIIFVVYTMLPFNMRDAIIASVLTSSSHTIVLSVCLSATPG---GKEHLVWQIL

1CJK ------

ADCY5 -IWWTVFFIYTIYTLLPVRMRAAVLSGVLLSALHLAIA---L-RTNA---QDQFLLKQLV

ADCY9 SRGLLHGCIHAIGVHLFVMSQVRSRSTFLKVGQSIMHGKDLEVEKALKERMIHSVMPRII

ADCY3 ANVFLYLCAIAVGIMSYYMADRKHRKAFLEARQSLEVKMNLEEQSQQQENLMLSILPKHV

ADCY8 AQAVLFMCMNTAGIFISYLSDRAQRQAFLETRRCVEARLRLETENQRQERLVLSVLPRFV

ADCY1 ANALLFVGVNMYGVFVRILTERSQRKAFLQARSCIEDRLRLEDENEKQERLLMSLLPRNV

ADCY4 ANAVLFLCGNVAGVYHKALMERALRATFREALSSLHSRRRLDTEKKHQEHLLLSILPAYL

ADCY7 ANAVIFLCGNLTGAFHKHQMQDASRDLFTYTVKCIQIRRKLRIEKRQQENLLLSVLPAHI

ADCY6 ANVLLFLCTNVIGICTHYPAEVSQRQAFQETRGYIQARLHLQHENRQQERLLLSVLPQHV

ADCY2 ANVIIFICGNLAGAYHKHLMELALQQTYQDTCNCIKSRIKLEFEKRQQERLLLSLLPAHI

1CJK ------

ADCY5 SNVLIFSCTNIVGVCTHYPAEVSQRQAFQETRECIQARLHSQRENQQQERLLLSVLPRHV

ADCY9 ADDLMKQGDEESENSVKRHATSSPKNRKKKSSIQKAPIAFRPFKMQQIEEVSILFADIVG

ADCY3 ADEMLKDMKKDESQ------KDQQQFNTMYMYRHENVSILFADIVG

ADCY8 VLEMINDMTNVEDE------HLQHQFHRIYIHRYENVSILFADVKG

ADCY1 AMEMKEDFLKP------PERIFHKIYIQRHDNVSILFADIVG

ADCY4 AREMKAEIMARLQA------GQGSRPESTNNFHSLYVKRHQGVSVLYADIVG

ADCY7 SMGMKLAIIERLKE------HGDRRCMPDNNFHSLYVKRHQNVSILYADIVG

ADCY6 AMEMKEDINTK------KEDMMFHKIYIQKHDNVSILFADIEG

ADCY2 AMEMKAEIIQRLQG------PKAGQMENTNNFHNLYVKRHTNVSILYADIVG

1CJK -MEMKADINAK------QEDMMFHKIYIQKHDNVSILFADIEG

ADCY5 AMEMKADINAK------QEDMMFHKIYIQKHDNVSILFADIEG

: *. : : : **:*:**: *

ADCY9 FTKMSANKSAHALVGLLNDLFGRFDRLCEETKCEKISTLGDCYYCVAGCPEPRADHAYCC

ADCY3 FTQLSSACSAQELVKLLNELFARFDKLAAKYHQLRIKILGDCYYCICGLPDYREDHAVCS

ADCY8 FTNLSTTLSAQELVRMLNELFARFDRLAHEHHCLRIKILGDCYYCVSGLPEPRQDHAHCC

ADCY1 FTGLASQCTAQELVKLLNELFGKFDELATENHCRRIKILGDCYYCVSGLTQPKTDHAHCC

ADCY4 FTRLASECSPKELVLMLNELFGKFDQIAKEHECMRIKILGDCYYCVSGLPLSLPDHAINC

ADCY7 FTQLASDCSPKELVVVLNELFGKFDQIAKANECMRIKILGDCYYCVSGLPVSLPTHARNC

ADCY6 FTSLASQCTAQELVMTLNELFARFDKLAAENHCLRIKILGDCYYCVSGLPEARADHAHCC

ADCY2 FTRLASDCSPGELVHMLNELFGKFDQIAKENECMRIKILGDCYYCVSGLPISLPNHAKNC

1CJK FTSLASQCTAQELVMTLNELFARFDKLAAENHCLRIKILGDCYYCVSGLPEARADHAHCC

ADCY5 FTSLASQCTAQELVMTLNELFARFDKLAAENHCLRIKILGDCYYCVSGLPEARADHAHCC

** ::: : ** **:**.:**.:. . :*. *******:.* ** .

ADCY9 IEMGLGMIKAIEQFCQEKKEMVNMRVGVHTGTVLCGILGMRRFKFDVWSNDVNLANLMEQ

ADCY3 ILMGLAMVEAISYVREKTKTGVDMRVGVHTGTVLGGVLGQKRWQYDVWSTDVTVANKMEA

ADCY8 VEMGLSMIKTIRYVRSRTKHDVDMRIGIHSGSVLCGVLGLRKWQFDVWSWDVDIANKLES

ADCY1 VEMGLDMIDTITSVAEATEVDLNMRVGLHTGRVLCGVLGLRKWQYDVWSNDVTLANVMEA

ADCY4 VRMGLDMCRAIRKLRAATGVDINMRVGVHSGSVLCGVIGLQKWQYDVWSHDVTLANHMEA

ADCY7 VKMGLDMCQAIKQVREATGVDINMRVGIHSGNVLCGVIGLRKWQYDVWSHDVSLANRMEA

ADCY6 VEMGVDMIEAISLVREVTGVNVNMRVGIHSGRVHCGVLGLRKWQFDVWSNDVTLANHMEA

ADCY2 VKMGLDMCEAIKKVRDATGVDINMRVGVHSGNVLCGVIGLQKWQYDVWSHDVTLANHMEA

1CJK VEMGMDMIEAISLVREMTGVNVNMRVGIHSGRVHCGVLGLRKWQFDVWSNDVTLANHMEA

ADCY5 VEMGMDMIEAISLVREVTGVNVNMRVGIHSGRVHCGVLGLRKWQFDVWSNDVTLANHMEA

: **: * :* . . ::**:*:*:* * *::* :::::**** ** :** :*

ADCY9 LGVAGKVHISEATAKYLDDRYEMEDGKVIERLGQSVVADQLKGLKTYLISGQRAKESRCS

ADCY3 GGIPGRVHISQSTMDCLKGEFDVEPGDGGSRCDY----LEEKGIETYLIIASKPEVKKTA

ADCY8 GGIPGRIHISKATLDCLNGDYNVEEGHGKERNEF----LRKHNIETYLIKQPEDSLLSLP

ADCY1 AGLPGKVHITKTTLACLNGDYEVEPGYGHERNSF----LKTHNIETFFIVPSHRRKIF-P

ADCY4 GGVPGRVHITGATLALLAGAYAVEDAGMEHRDPY----LRELGEPTYLVIDPRAEEEDEK

ADCY7 AGVPGRVHITEATLKHLDKAYEVEDGHGQQRDPY----LKEMNIRTYLVIDPRSQQPPPP

ADCY6 GGRAGRIHITRATLQYLNGDYEVEPGRGGERNAY----LKEQHIETFLILGASQKRKEEK

ADCY2 GGVPGRVHISSVTLEHLNGAYKVEEGDGDIRDPY----LKQHLVKTYFVINPKGERRSPQ

1CJK GGKAGRIHITKATLSYLNGDYEVEPGCGGERNAY----LKEHSIETFLILRCTQKRKEEK

ADCY5 GGKAGRIHITKATLNYLNGDYEVEPGCGGERNAY----LKEHSIETFLILRCTQKRKEEK

* *::**: * * : :* . * . *:::

ADCY9 CAEALLSGFEVIDGSQVSSGPRGQGTASSGNVSDLAQTVKTFDNLKTCP----SCGITFA

ADCY3 TQNG------LNGSALPN------GAPASSKSSSP------ALIET

ADCY8 EDIV------KE------SVSSSD---RR----NSGATFT

ADCY1 GLIL------SD------IKPAKRMKFKTVCYLLVQLMHC

ADCY4 GTAG------GL------LSSLEGLKMRP----SLLMTRY

ADCY7 SQHL------PR------PKGDAALKMRA----SVRMTRY

ADCY6 AMLA------KL------QR------TR----ANSMEGL

ADCY2 HLFR------PR------HT-LDGAKMRA----SVRMTRY

1CJK AMIA------KR------S------

ADCY5 AMIA------KM------NR------QR----TNSIGHN

ADCY9 PKSEAGAEGGAPQNGCQDEHKNSTKASGGPNPKTQNGLLSPPQEEKLTNSQTSLCEILQE

ADCY3 KEPNGS----AHSSGSTSEK-PEEQDAQADNPSFP-----NPRRR------

ADCY8 EGSWSPELPFDNIVGKQNTLAALTRNSINLLPNHL------AQA------

ADCY1 RKMFKAEIPFSNVMTCEDDDKRRA------LRT------ASEK------

ADCY4 LESWGAAKPFAHLSHGDSPVSTSTPLPEKTLASFS------TQ------

ADCY7 LESWGAARPFAHLNHRESVSSGETHVPNGRRPKSV------PQRH------

ADCY6 MPRWVPDRAFSRTKDSK----AFRQMGIDD------

ADCY2 LESWGAAKPFAHLHHRDSMTTENGKISTTDVPMGQ------HN------

1CJK ------

ADCY5 PPHWGAERPFYNHLGGNQVSKEMKRMG------

ADCY9 KGRWAGVSLDQSALLPLRFKNIREKTDAHFVDVIKEDSLM-KDYFFKPPINQFSLNFLDQ

ADCY3 ---LRLQD---LADRVVDASEDEHELNQLLNEALLERES--AQVVKKRNTFLLSMRFMDP

ADCY8 ------LHVQSGPEEINKRIEHTIDLRS---GDKLRREHIKPFSLMFKDS

ADCY1 ---LRNRS---SFSTNVVYTTPGTRVNRYISRLLEAR----QTELEMADLNFFTLKYKHV

ADCY4 ---W-SLD---RSRTPRGLDDELDTGDAKFFQVIEQLNSQK-QWKQSKDFNPLTLYFREK

ADCY7 ---RRTPD---RSMSPKGR-SEDDSYDDEMLSAIEGLSSTRPCCSKSDDFYTFGSIFLEK

ADCY6 ----SSKD---NRGTQDAL-NPEDEVDEFLSRAIDAR--SI-DQLRKDHVRRFLLTFQRE

ADCY2 ---FQNRT---LRTKSQKK-RFEEELNERMIQAIDGINAQK-QWLKSEDIQRISLLFYNK

1CJK ------

ADCY5 ---FEDPK---DKNAQESA-NPEDEVDEFLGRAIDAR--SI-DRLRSEHVRKFLLTFREP

ADCY9 ELERSYRTSYQEEVIKNSPVKTFASPTFSSLLDVFLSTTVFLTLSTTCFLKYEAATVPPP

ADCY3 EMETRYSVEKEKQSGAAFSC------SCVVLL------CTALVEILIDPWL

ADCY8 SLEHKYSQMRDEVFKSNLVC------AFIVLL------FITAIQSLLPSSR

ADCY1 EREQKYHQLQDEYFTSAVVL------TLILAA------LFGLVYLLIFPQS

ADCY4 EMEKEYRLSAIPAFKYYEAC------TFLVFL------SNFIIQMLVTNRP

ADCY7 GFEREYRLAPIPRARHDFAC------ASLIFV------CILLVHVLLMPRT

ADCY6 DLEKKYSRKVDPRFGAYVAC------ALLVFC------FICFIQLLIFPHS

ADCY2 VLEKEYRATALPAFKYYVTC------ACLIFF------CIFIVQILVLPKT

1CJK ------

ADCY5 DLEKKYSKQVDDRFGAYVAC------ASLVFL------FICFVQITIVPHS

ADCY9 PA-ALAVFSAALLLEVLSLAVSIRMVFFLEDVM-ACTKRLLEWIAGWLPR------HC-

ADCY3 MT-NYVTFMVGE-ILLLIL-----TICSLAAIFPRAFPKKLVA------FSTWIDRTRW

ADCY8 VMPMTIQFSILIMLHSALV------LITTAEDY-KCLPLILRKTCCWINE------TYL

ADCY1 VV-VLLL------LVF-----CICFLVA----CVLYLHITRVQCFP---GCL--TIQ

ADCY4 PA-LAITYSITFLLFLLIL-----FVCFSEDLM-RCVLKGP-KMLHWLPALSGLVATRPG

ADCY7 AA-LGVSFGLVACVLGLVL-----GLCFATKFS-RCCPARG-TLCT----ISERVETQPL

ADCY6 TL-MLGIYASIFLLLLITV-----LIC---AVY-SCGSLFP-KALQRLSR---SIVRSRA

ADCY2 SV-LGISFGAAFLLLAFIL-----FVCFAGQLL-QCSKKAS-PLLMWLLKSSGIIANRPW

1CJK ------

ADCY5 IF-MLSFYLTCSLLLTLVV-----F---VSVIY-SCVKLFP-SPLQTLS--RKI-VRSKM

ADCY9 ----IGAILVSL------PALAVYSHVTSE------YETNIHFPVF

ADCY3 ARNTWAMLAIFILVMANVVDML------SCLQYYTGPSNA------TAG

ADCY8 ARNVIIFASILINFLGAILNILWCDFDKSI-PLKN------LTF---

ADCY1 IRTVLCIFIVVLIYSVA------QGCVVGCLPWAWSSKPN------SSLVVLSSG

ADCY4 LRIALGTATILLVFAMAITSLFFFPTSSDCP---FQAPNVSSMI------SNLSWELP

ADCY7 LRLTLAVLTIGSLLTVAIINLPLMPFQVPE------LPVG

ADCY6 HSTAVGIFSVLLVFTSAIANMF-TCNHTPIRSCAARMLNLTPADITACHLQQ--LNYSLG

ADCY2 PRISLTIITTAIILMMAVFNMFFLSDSEETIPPTANTTNTS------FSAS

1CJK ------

ADCY5 NSTLVGVFTITLVFLAAFVNMF-TCNSRDLLGCLAQEHNISASQVNACHVAESAVNYSLG

ADCY9 ------TGSAALIAVVHYCNFCQLSSWM------RSSLATVVGAGPLLLLY

ADCY3 ------METEGSCLENPKYYNYVAVLSLIATIMLVQVSHMVKLTLMLLVAGAVATINL

ADCY8 ------NSSAVFTDICSYPEYFVFTGVLAMVTCAVFLRLNSVLKLAVLLIMIAIYALLTE

ADCY1 G-----QRTALPTLPCESTHHALLCCLVGTLPLAIFFRVSSLPKMILLSGLTTSYILVLE

ADCY4 ------GS-LPLISVPYSMHCCTLGFLSCSLFLHMSFELKLLLLLLWLAASCSLFL

ADCY7 NETGLLAASSKTRALCEPLPYYTCSCVLGFIACSVFLRMSLEPKVVLLTVALVAYLVLFN

ADCY6 LDAPLC---EGTMPTCSFPEYFIGNMLLSLLASSVFLHISSIGKLAMIFVLGLIYLVLLL

ADCY2 N-NQVA---ILRAQNLFFLPYFIYSCILGLISCSVFLRVNYELKMLIMMVALVGYNTI-L

1CJK ------

ADCY5 DEQGFC---GSPWPNCNFPEYFTYSVLLSLLACSVFLQISCIGKLVLMLAIELIYVLI-V

ADCY9 VSLCPDSSVLTSPLDAVQNFSS------ERNPCNSSVPRDLRRPASLIGQEV

ADCY3 --YAWRP-----VFDEYDHKRFREHDLPMVALEQMQGFNPGLNGTDRLPLVPSKY---SM

ADCY8 TVYA------GLFLRYDNLNH------SGEDFLGTKE---VS

ADCY1 LSG-YT------RT------GGGAVSGRSY---EP

ADCY4 HSHAWLSECLIVRL------YLGPLDSRPGVLKEPKL---MG

ADCY7 LSPCWQWDCCGQGLG------N------LTKPNGTTSGTPSCSWKDLKT---MT

ADCY6 LGPPATIFDNYDLLLGVHGLAS------SNETFDGLDCPA-AGRVALKY---MT

ADCY2 LH------THAHVLGDYSQVLF------E------RPGIWKDLKT---MG

1CJK ------

ADCY5 EVPGVTLFDNADLLVTANAIDF------FNN--GTSQCPEHATKVALKV---VT

ADCY9 VLVFFLLLLLVWFLNREFEVSYRLHYHGDVEADLHRTKIQSMRDQADWLLRNIIPYHVAE

ADCY3 TVMVFLMMLSFYYFSRHVEKLARTLFLWKIEVHDQKERVYEMRRWNEALVTNMLPEHVAR

ADCY8 LLLMAMFLLAVFYHGQQLEYTARLDFLWRVQAKEEINEMKELREHNENMLRNILPSHVAR

ADCY1 IVAILLFSCALALHARQVDIRLRLDYLWAAQAEEEREDMEKVKLDNRRILFNLLPAHVAQ

ADCY4 AISFFIFFFTLLVLARQNEYYCRLDFLWKKKLRQEREETETMENLTRLLLENVLPAHVAP

ADCY7 NFYLVLFYITLLTLSRQIDYYCRLDCLWKKKFKKEHEEFETMENVNRLLLENVLPAHVAA

ADCY6 PVILLVFALALYLHAQQVESTARLDFLWKLQATGEKEEMEELQAYNRRLLHNILPKDVAA

ADCY2 SVSLSIFFITLLVLGRQNEYYCRLDFLWKNKFKKEREEIETMENLNRVLLENVLPAHVAE

1CJK ------

ADCY5 PIIISVFVLALYLHAQQVESTARLDFLWKLQATEEKEEMEELQAYNRRLLHNILPKDVAA

ADCY9 QLK-----VSQTYSKNHDSGGVIFASIVNFSEFYEEN--YEGGKECYRVLNELIGDFDEL

ADCY3 HFLGSKKRDEELYSQTYDEIGVMFASLPNFADFYTEESINNGGIECLRFLNEIISDFDSL

ADCY8 HFLEKDRDNEELYSQSYDAVGVMFASIPGFADFYSQTEMNNQGVECLRLLNEIIADFDEL

ADCY1 HFLMSNPRNMDLYYQSYSQVGVMFASIPNFNDFYIELDGNNMGVECLRLLNEIIADFDEL

ADCY4 QFIGQNRRNEDLYHQSYECVCVLFASVPDFKEFYSESNINHEGLECLRLLNEIIADFDEL

ADCY7 HFIGDK-LNEDWYHQSYDCVCVMFASVPDFKVFYTECDVNKEGLECLRLLNEIIADFDEL

ADCY6 HFLARERRNDELYYQSCECVAVMFASIANFSEFYVELEANNEGVECLRLLNEIIADFDEI

ADCY2 HFLARSLKNEELYHQSYDCVCVMFASIPDFKEFYTESDVNKEGLECLRLLNEIIADFDDL

1CJK ------LKNEELYHQSYDCVCVMFASIPDFKEFYTESDVNKEGLECLRLLNEIIADFDDL

ADCY5 HFLARERRNDELYYQSCECVAVMFASIANFSEFYVELEANNEGVECLRLLNEIIADFDEI

: * :. . *:***: .* ** : . * ** *.***:*.***.:

ADCY9 LSKPDYSSIEKIKTIGATYMAASGLNTAQA------QDGSHPQEHLQILFEFAKE

ADCY3 LDNPKFRVITKIKTIGSTYMAASGVTPDVNTNGFASSNKEDKSERERWQHLADLADFALA

ADCY8 LGEDRFQDIEKIKTIGSTYMAVSGLSPEK------QQCEDKWGHLCALADFSLA

ADCY1 MEKDFYKDIEKIKTIGSTYMAAVGLAPTSG------TKAKK---SISSHLSTLADFAIE

ADCY4 LSKPKFSGVEKIKTIGSTYMAATGLNATSG------QDAQQDAERSCSHLGTMVEFAVA

ADCY7 LLKPKFSGVEKIKTIGSTYMAAAGLSVASG------HENQELERQHAHIGVMVEFSIA

ADCY6 ISEERFRQLEKIKTIGSTYMAASGLNASTY------DQ------VGRSHITALADYAMR

ADCY2 LSKPKFSGVEKIKTIGSTYMAATGLSAVPS------QEHSQEPERQYMHIGTMVEFAFA

1CJK LSKPKFSGVEKIKTIGSTYMAATGLSAIPS------QEHAQEPERQYMHIGTMVEFAYA

ADCY5 ISEDRFRQLEKIKTIGSTYMAASGLNDSTY------DK------VGKTHIKALADFAMK

: : : : ******:****. *: *: : :::

ADCY9 MMRVVDDFNNNMLWFNFKLRVGFNHGPLTAGVIGTTKLLYDIWGDTVNIASRMDTTGVEC

ADCY3 MKDTLTNINN-QSFNNFMLRIGMNKGGVLAGVIGARKPHYDIWGNTVNVASRMESTGVMG

ADCY8 LTESIQEINK-HSFNNFELRIGISHGSVVAGVIGAKKPQYDIWGKTVNLASRMDSTGVSG

ADCY1 MFDVLDEINY-QSYNDFVLRVGINVGPVVAGVIGARRPQYDIWGNTVNVASRMDSTGVQG

ADCY4 LGSKLDVINK-HSFNNFRLRVGLNHGPVVAGVIGAQKPQYDIWGNTVNVASRMESTGVLG

ADCY7 LMSKLDGINR-HSFNSFRLRVGINHGPVIAGVIGARKPQYDIWGNTVNVASRMESTGELG

ADCY6 LMEQMKHINE-HSFNNFQMKIGLNMGPVVAGVIGARKPQYDIWGNTVNVSSRMDSTGVPD

ADCY2 LVGKLDAINK-HSFNDFKLRVGINHGPVIAGVIGAQKPQYDIWGNTVNVASRMDSTGVLD

1CJK LVGKLDAINK-HSFNDFKLRVGINHGPVIAGVIGAQKPQYDIWGNTVNVASRMDSTGVLD

ADCY5 LMDQMKYINE-HSFNNFQMKIGLNIGPVVAGVIGARKPQYDIWGNTVNVASRMDSTGVPD

: : :* : .* :::*:. * : *****: : *****.***::***::**

ADCY9 RIQVSEESYRVLSKMGYDFDYRGTVNVKGK----GQMKTYLYPKCTDHRVIPQHQLSISP

ADCY3 NIQVVEETQVILREYGFRFVRRGPIFVKGK----GELLTFFLKGRDKLATFPNGPSVTLP

ADCY8 RIQVPEETYLILKDQGFAFDYRGEIYVKGISEQEGKIKTYFLLGRVQPNPFILPPRR-LP

ADCY1 RIQVTEEVHRLLRRCPYHFVCRGKVSVKGK----GEMLTYFLEGRTDGNGSQIR------

ADCY4 KIQVTEETAWALQSLGYTCYSRGVIKVKGK----GQLCTYFLNTDLTRTGPPSA------

ADCY7 KIQVTEETCTILQGLGYSCECRGLINVKGK----GELRTYFVCTDTAKFQGLGL------

ADCY6 RIQVTTDLYQVLAAKGYQLECRGVVKVKGK----GEMTTYFLNGGPSS------

ADCY2 KIQVTEETSLVLQTLGYTCTCRGIINVKGK----GDLKTYFVNTEMSRSLSQSN------

1CJK KIQVTEETSLILQTLGYTCTCRGIINVKGK----GDLKTYFVNTEMSR------

ADCY5 RIQVTTDMYQVLAANTYQLECRGVVKVKGK----GEMMTYFLNGGPPLS------

.*** : * : ** : *** *.: *::

ADCY9 DI------RVQVDGSIGRSPTDEIANLVPSVQYVDKTSLGSDSSTQAKDAHLSPKRPWKE

ADCY3 HQVVDNS------

ADCY8 GQYSLAAVVLGLVQSLNRQRQKQLLNENNNTGIIKGH------YNRRTL--LSPSGT

ADCY1 ------SLGLDRKMCPFGRAGLQGRRPPVCPMPGV------SVRAGLPPHSPGQY

ADCY4 ------TLG------

ADCY7 ------N------

ADCY6 ------

ADCY2 ------VAS------

1CJK ------

ADCY5 ------

ADCY9 PVKAEERGRFGKAIEKDDCDETGIEEANELTKLNVSKSV

ADCY3 ------

ADCY8 EPGAQAEGT-----DKSDLP------

ADCY1 LPSA-AAGK-----EA------

ADCY4 ------

ADCY7 ------

ADCY6 ------

ADCY2 ------

1CJK ------

ADCY5 ------

Supplementary Table 1. Prediction of S-nitrosylation sites using GPS-NO3.0 and NSO 1.0 softwares for AC5.

GPS-NO 3.0 software prediction

PEPTIDE / POSITION / AROC
SCORE / CUTOFF / CLUSTER
SVFLQISCIGKLVLM / 929 / 4.402 / 2.443 / Cluster B
FNNGTSQCPEHATKV / 977 / 2.647 / 2.443 / Cluster B
ANNEGVECLRLLNEI / 1096 / 2.815 / 2.443 / Cluster B

NSO 1.0 software prediction

Protein Name / Locations / Nitrosylation Sites / The matched MDD-clustered Motifs
ADCY5 / 208 / GPGAVLSLGACCLALLQIFRS /
ADCY5 / 209 / PGAVLSLGACCLALLQIFRSK /
ADCY5 / 395 / IFSCTNIVGVCTHYPAEVSQR /
ADCY5 / 414 / QRQAFQETRECIQARLHSQRE /
ADCY5 / 510 / RFDKLAAENHCLRIKILGDCY /
ADCY5 / 519 / HCLRIKILGDCYYCVSGLPEA /
ADCY5 / 522 / RIKILGDCYYCVSGLPEARAD /
ADCY5 / 536 / LPEARADHAHCCVEMGMDMIE /
ADCY5 / 537 / PEARADHAHCCVEMGMDMIEA /
ADCY5 / 572 / RVGIHSGRVHCGVLGLRKWQF /
ADCY5 / 624 / LNGDYEVEPGCGGERNAYLKE /
ADCY5 / 645 / HSIETFLILRCTQKRKEEKAM /
ADCY5 / 767 / VDDRFGAYVACASLVFLFICF /
ADCY5 / 796 / SIFMLSFYLTCSLLLTLVVFV /
ADCY5 / 812 / LVVFVSVIYSCVKLFPSPLQT /
ADCY5 / 856 / FLAAFVNMFTCNSRDLLGCLA /
ADCY5 / 864 / FTCNSRDLLGCLAQEHNISAS /
ADCY5 / 879 / HNISASQVNACHVAESAVNYS /
ADCY5 / 977 / IDFFNNGTSQCPEHATKVALK /
ADCY5 / 1069 / NDELYYQSCECVAVMFASIAN /
ADCY5 / 1096 / ELEANNEGVECLRLLNEIIAD /
ADCY5 / 1237 / VLAANTYQLECRGVVKVKGKG /

Supplementary Table 2. Prediction of S-nitrosylation sites using GPS-NO3.0 and NSO 1.0 softwares for AC6.

GPS-NO 3.0 software prediction

PEPTIDE / POSITION / AROC
SCORE / CUTOFF / CLUSTER
VPRSGRSCWRRLVQV / 118 / 3.054 / 2.443 / Cluster B
AYVALLACAAALFVG / 186 / 2.848 / 2.443 / Cluster B
NHTPIRSCAARMLNL / 772 / 2.864 / 2.443 / Cluster B
ANNEGVECLRLLNEI / 1004 / 2.815 / 2.443 / Cluster B

NSO 1.0 software prediction

Protein Name / Locations / Nitrosylation Sites / The matched MDD-clustered Motifs
ADCY6 / 45 / GGFCTPRYMSCLRDAEPPSPT /
ADCY6 / 297 / QLGANVLLFLCTNVIGICTHY /
ADCY6 / 304 / LFLCTNVIGICTHYPAEVSQR /
ADCY6 / 419 / RFDKLAAENHCLRIKILGDCY /
ADCY6 / 428 / HCLRIKILGDCYYCVSGLPEA /
ADCY6 / 431 / RIKILGDCYYCVSGLPEARAD /
ADCY6 / 445 / LPEARADHAHCCVEMGVDMIE /
ADCY6 / 446 / PEARADHAHCCVEMGVDMIEA /
ADCY6 / 481 / RVGIHSGRVHCGVLGLRKWQF /
ADCY6 / 674 / VDPRFGAYVACALLVFCFICF /
ADCY6 / 714 / FLLLLITVLICAVYSCGSLFP /
ADCY6 / 719 / ITVLICAVYSCGSLFPKALQR /
ADCY6 / 763 / FTSAIANMFTCNHTPIRSCAA /
ADCY6 / 771 / FTCNHTPIRSCAARMLNLTPA /
ADCY6 / 802 / NYSLGLDAPLCEGTMPTCSFP /
ADCY6 / 809 / APLCEGTMPTCSFPEYFIGNM /
ADCY6 / 885 / SSNETFDGLDCPAAGRVALKY /
ADCY6 / 976 / NDELYYQSCECVAVMFASIAN /
ADCY6 / 1003 / ELEANNEGVECLRLLNEIIAD /
ADCY6 / 1144 / VLAAKGYQLECRGVVKVKGKG /

Supplementary Table 3. Prediction of S-nitrosylation sites using GPS-NO3.0 and NSO 1.0 softwares for AC9.

GPS-NO 3.0 software prediction

PEPTIDE / POSITION / AROC
SCORE / CUTOFF / CLUSTER
KYSISSSCSSSGDSG / 54 / 1.607 / 1.484 / Cluster A
AKESRCSCAEALLSG / 583 / 2.739 / 2.443 / Cluster B
FLEDVMACTKRLLEW / 851 / 3.451 / 2.443 / Cluster B
AGWLPRHCIGAILVS / 867 / 3.092 / 2.443 / Cluster B

NSO 1.0 software prediction

Protein Name / Locations / Nitrosylation Sites / The matched MDD-clustered Motifs
ADCY9 / 19 / LLHHHSTEVSCDSSGDSNSVR /
ADCY9 / 46 / QLSSNSHPKHCKYSISSSCSS /
ADCY9 / 104 / KFDSVNLEEACLERCFPQTQR /
ADCY9 / 127 / RYALFYIGFACLLWSIYFAVH /
ADCY9 / 151 / RLIVMVAPALCFLLVCVGFFL /
ADCY9 / 227 / CLSQVGSFSMCIEVLFLLYTV /
ADCY9 / 247 / VMHLPLYLSLCLGVAYSVLFE /
ADCY9 / 268 / TFGYHFRDEACFPSPGAGALH /
ADCY9 / 290 / ELLSRGLLHGCIHAIGVHLFV /
ADCY9 / 430 / NDLFGRFDRLCEETKCEKIST /
ADCY9 / 447 / KISTLGDCYYCVAGCPEPRAD /
ADCY9 / 451 / LGDCYYCVAGCPEPRADHAYC /
ADCY9 / 461 / CPEPRADHAYCCIEMGLGMIK /
ADCY9 / 462 / PEPRADHAYCCIEMGLGMIKA /
ADCY9 / 477 / LGMIKAIEQFCQEKKEMVNMR /
ADCY9 / 497 / RVGVHTGTVLCGILGMRRFKF /
ADCY9 / 581 / ISGQRAKESRCSCAEALLSGF /
ADCY9 / 583 / GQRAKESRCSCAEALLSGFEV /
ADCY9 / 630 / TVKTFDNLKTCPSCGITFAPK /
ADCY9 / 633 / TFDNLKTCPSCGITFAPKSEA /
ADCY9 / 654 / GAEGGAPQNGCQDEHKNSTKA /
ADCY9 / 693 / EKLTNSQTSLCEILQEKGRWA /
ADCY9 / 867 / EWIAGWLPRHCIGAILVSLPA /
ADCY9 / 940 / GPLLLLYVSLCPDSSVLTSPL /
ADCY9 / 963 / VQNFSSERNPCNSSVPRDLRR /
ADCY9 / 1081 / YEENYEGGKECYRVLNELIGD /
ADCY9 / 1204 / ASRMDTTGVECRIQVSEESYR /
ADCY9 / 1245 / GQMKTYLYPKCTDHRVIPQHQ /
ADCY9 / 1333 / RFGKAIEKDDCDETGIEEANE /

Supplementary Table 4. Prediction of S-nitrosylation sites using GPS-NO3.0 and NSO 1.0 softwares for Gαs

GPS-NO 3.0 software prediction

PEPTIDE / POSITION / SCORE / CUTOFF / CLUSTER
*****MGCLGNSKTE / 3 / 4.462 / 2.443 / Cluster B

NSO 1.0 software prediction

Protein Name / Locations / Nitrosylation Sites / The matched MDD-clustered Motifs
G-ALPHA-S / 162 / ALWEDEGVRACYERSNEYQLI /
G-ALPHA-S / 174 / ERSNEYQLIDCAQYFLDKIDV /
G-ALPHA-S / 200 / YVPSDQDLLRCRVLTSGIFET /
G-ALPHA-S / 237 / QRDERRKWIQCFNDVTAIIFV /
G-ALPHA-S / 359 / STASGDGRHYCYPHFTCAVDT /
G-ALPHA-S / 365 / GRHYCYPHFTCAVDTENIRRV /
G-ALPHA-S / 379 / TENIRRVFNDCRDIIQRMHLR /
Sl. No. / Compound / Docking Score / glide energy
1 / ZINC85642736 / -10.186 / -47.481
2 / ZINC67912726 / -8.357 / -44.409
3 / 4f / -8.229 / -53.556
4 / 4b / -8.153 / -48.028
5 / ZINC43552801 / -8.074 / -30.569
6 / ZINC06186553 / -7.994 / -65.844
7 / ZINC25725790 / -7.855 / -54.566
8 / 4e / -7.861 / -62.487
9 / 4c / -7.572 / -62.996
10 / 4d / -7.560 / -59.862
11 / 4a / -7.548 / -50.430
12 / ZINC04349857 / -7.514 / -43.101
13 / 12b / -7.557 / -46.593
14 / ZINC27555316 / -7.477 / -39.468
15 / 9a / -7.679 / -45.641

Supplementary Table 5. Virtual ligand screening using Glide algorithm in Schroedinger software.