Elucidation of Agonist and Antagonist Dynamic Binding ...

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Elucidation of Agonist and Antagonist Dynamic Binding Patterns in ER- by Integration of Molecular Docking, Molecular Dynamics Simulations and Quantum Mechanical Calculations

Sugunadevi Sakkiah, Chandrabose Selvaraj , Wenjing Guo, Jie Liu, Weigong Ge, Tucker A. Patterson and Huixiao Hong *

Division of Bioinformatics and Biostatistics, National Center for Toxicological Research, U.S. Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA; Suguna.Sakkiah@fda. (S.S.); selnikraj@ (C.S.); Wenjing.Guo@fda. (W.G.); Jie.Liu1@fda. (J.L.); Weigong.Ge@fda. (W.G.); Tucker.Patterson@fda. (T.A.P.) * Correspondence: Huixiao.Hong@fda.; Tel.: +1-870-543-7296 Current affiliation: Department of Bioinformatics, Alagappa University, Karaikudi 630 003, Tamil Nadu, India.

Citation: Sakkiah, S.; Selvaraj, C.; Guo, W.; Liu, J.; Ge, W.; Patterson, T.A.; Hong, H. Elucidation of Agonist and Antagonist Dynamic Binding Patterns in ER- by Integration of Molecular Docking, Molecular Dynamics Simulations and Quantum Mechanical Calculations. Int. J. Mol. Sci. 2021, 22, 9371. 10.3390/ijms22179371

Abstract: Estrogen receptor alpha (ER) is a ligand-dependent transcriptional factor in the nuclear receptor superfamily. Many structures of ER bound with agonists and antagonists have been determined. However, the dynamic binding patterns of agonists and antagonists in the binding site of ER remains unclear. Therefore, we performed molecular docking, molecular dynamics (MD) simulations, and quantum mechanical calculations to elucidate agonist and antagonist dynamic binding patterns in ER. 17-estradiol (E2) and 4-hydroxytamoxifen (OHT) were docked in the ligand binding pockets of the agonist and antagonist bound ER. The best complex conformations from molecular docking were subjected to 100 nanosecond MD simulations. Hierarchical clustering was conducted to group the structures in the trajectory from MD simulations. The representative structure from each cluster was selected to calculate the binding interaction energy value for elucidation of the dynamic binding patterns of agonists and antagonists in the binding site of ER. The binding interaction energy analysis revealed that OHT binds ER more tightly in the antagonist conformer, while E2 prefers the agonist conformer. The results may help identify ER antagonists as drug candidates and facilitate risk assessment of chemicals through ER-mediated responses.

Academic Editor: Gerard Pujadas

Received: 27 May 2021 Accepted: 27 August 2021 Published: 29 August 2021

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Copyright: ? 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// licenses/by/ 4.0/).

Keywords: molecular docking; molecular dynamics simulations; quantum mechanical calculations; estrogen receptor; dynamic binding pattern

1. Introduction Estrogen receptor (ER) is one of the important targets of drugs and endocrine disrupt-

ing chemicals in the endocrine system [1]. It is a ligand-dependent transcriptional factor in the steroid type 1 nuclear receptor family [2]. ER plays a major role in various biological functions such as bone modeling, reproductive system, cardiovascular system, metabolism, and cell proliferation [3]. ER is an extensively studied target among the endocrine receptors. There are two major ER isoforms, ER and ER. Like other nuclear receptors, ER consists of three distinct domains: N-terminal domain (residue 1?180), DNA binding domain (residue 181?263), and C-terminal domain or ligand binding domain (LBD, residue 303?552) (Figure 1). The activation function domain 1 (AF1) is present in the N-terminal domain and plays a major role in the protein?protein interaction [4,5]. The mitogen-activated protein (MAP) kinase pathway regulates the activity of AF1 through the growth factors [6]. The LBD is composed of twelve helices and two antiparallel -sheets which are arranged as a three-layer antiparallel helical sandwich [5,7]. The first layer is formed by helices 1 to 4 and 7, the middle layer is made up of helices 5, 6, 9 and 10 and the final layer is

Int. J. Mol. Sci. 2021, 22, 9371.



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Int. J. Mol. Sci. 2021, 22, 9371

through the growth factors [6]. The LBD is composed of twelve helices and two anti2poafr1-8

allel -sheets which are arranged as a three-layer antiparallel helical sandwich [5,7]. The

first layer is formed by helices 1 to 4 and 7, the middle layer is made up of helices 5, 6, 9

acnodm1p0oasnedd tohfehfeinliacel sla8y,earnids c1o1m[8p?o1s0e]d. Tofhheealcicteivsa8t,ioanndfu1n1c[t8io?n10d].oTmhaeinac2ti(vAatFi2o)nifnuLnBctDionis droemspaoinns2ib(lAeFfo2)r ibninLdBiDngisorfecsopfoancstoibrlse. fAorFb2iundndinegrgoofecsofcaocntoforrsm. AaFti2onuanldcehragnogese cdounefotromtha-e tbioinadlinchgaonfgaecdoumeptouthnedbinintdhiengligoafnadcobminpdoinugndpoincktehte(LligBaPn).dTbhiendcoinngfopromcakteito(nLaBl Pc)h.aTnhgee coofnAfoFr2mdaetitoenrmalinchesanthgeetoyfpeAsFo2f dbientedrimnginceosfatchteortsypwehsicohf pblianydianmg acjoofracrotolersinwahcitcivhaptilnagy oar minahjoibrirtionlge tihneatcatrivgaetignegnoesr oinfhEiRbi[t1in1g]. tThheetharinggete g(reenseidsuoef 2E6R4?[13012].)Trehgeiohninsgceon(rneescitdtuhee 2D6B4D? 3a0n2d) rLeBgDio.ns connect the DBD and LBD.

FFigiguurree11..TThheecacratrotononrerperperseesnetnattaiotinonofotfhtehedidffieffreernetndtodmomainaisnosfoEfRE.RL.BLPBiPs sishoshwonwin ignregerne;eAn;FA2 Fis2 sihsoswhonwinn einleeclteroctsrtoastitcatriecprerepsreensteanttiaotnio; nan; adnHd 1H21i2s isshsohwown nininmmagaegnetnataananddbblulueefoforraaccttivivee((aaggoonniisstt bboouunndd))aannddininaaccttivivee((aannttaaggoonniissttbboouunndd))ffoorrmmss,,rreessppeeccttiivveellyy..

TThheeHH1122aaccttssaassaammoolleeccuullaarrsswwiittcchhtthhaattttuurrnnssEERRaaccttiivviittyyoonnaannddooffff ddeeppeennddiinnggoonn tthheebbininddininggcchheemmicicaalsls[[1111??1133]]..TThheeAAFF11aannddAAFF22ppllaayyaammaajojorrrroolleeiinn tthhee ttrraannssccrriippttiioonnaall aacctitvivaatitoionnooffEERR[[1144]].. TThheeeessttrrooggeennicicccoommppoouunnddssbbininddinintthhee hhyyddrroopphhoobbicicppoocckkeettooff EERR LLBBDD. .TThheehhyyddrroopphhoobbicicppoocckkeettisisccoommppoosseeddooffMMeett334422ttooLLeeuu335544ooffHH33, ,TTrrpp338833ttooAArrgg339944 ooffHH66,,VVaall441188ttooLLeeuu442288ffrroommtthheepprreecceeddiinnggllooooppooffHH88,,MMeett551177ttooMMeett552288ooffHH1111,,LLeeuu553399 ttooHHiiss554477ooffHH1122,,aanndd LLeeuu440022 ttoo Leu 410 of S1//SS22hhaaiirrppiinn[[77]].. BBiinnddiinnggooff aannttiieessttrrooggeenniicc ccoommppoouunnddsstotoEERRinidnudcuecae Ha 1H21c2oncofonrfmoramtioatniaolncahlacnhgaensgbeys pblyacpinlagciHn1g2Hac1r2oasscrtohsesHth3eanHd3 Han11daHn1d1manodvinmgoHvi1n2gaHw1a2yafwroamy ftrhoemLBthPe. LDBuPe. tDoutheetoHt1h2e cHo1n2focromnafotiromnacthioangceh,atnhgeeA, tFh2e iAn Fth2einLtBhDe LisBdDisitsodrtiesdtoartnedd naontdsnuoittasbuleitafobrlebfionrdbiningdcionfgacctoofrasct[o1r5s].[1E5R].eEnRhaenncheasnacnesdarnedprreepsrseessseitssitfsunfucnticotniovniva iavavraioriuosuspaptahtwhwayasys[1[01,01,61,61,71]7.].UUnnddeersrtsatnanddininggththeeEERR ddyynnaammiicc bbininddininggppaattteterrnnsswwitihthaaggoonnisiststsaannddaannttaaggoonnisiststsisisccrruucciaiallffoorrddiissccoovveerryyooffEERR aaggoonniissttss aannddaannttaaggoonnisiststs. .DDyynnaammicicbbininddininggppaatttetrenrnrereppreresesenntstsththeefoformrmininggaannddbbrereaakkininggooffnnoonn-ccoovvaalelennttinintteerraacctitoionnssssuucchhaasshhyyddrrooggeennbboonnddininggaannddVVaannddeerrWWaaaallssiinntteerraaccttiioonnssbbeettwweeeenn aapprrootteeininaannddaalliiggaanndd,,aasswweellllaass ccoonnffoorrmmaattiioonnaallcchhaannggeessccaauusseeddbbyytthheebbiinnddiinngglliiggaanndd tthhrroouugghhoouuttaammoolleeccuulalarrddyynnaammicicss((MMDD))ssiimmuulalattioionn. .MMoorereththaann33550033DDssttrruuccttuurreessooff EERR bboouunnddwwiitthh vvaarriioouuss lliiggaannddssaarreeddeeppoosistietdedininthtehPerPorteoitneiDnaDtaatBaanBkan(PkD(PBD). BT)h.oTsheosstreuscttruurce-s taurreesuaserefuulsteofuulntdoeursntdanerdsttahnedsttrhuecstutrruacltcuhraanl cgheasndguees tdouaegtoonaisgtoannisdt aanntdagaonntaisgtobniinsdt biningdininthgeinERtheLEBRP. VLaBrPio. Vusarcioomuspcuotmatpiountaatlitoencahlntieqcuhensiqsuuecshsauscmh aoslemcuollaercudloacrkdioncgk[i1n8g?[2148]?, 2M4]D, MsiDmsuilmatuiolantsio[n25s?[3205]?,3p0r]e, pdricetdivicetimveomdeoldineglin[3g1[?3411?]4, 1a]n,danind vinitvroitrsotusdtuiedsiewsewreerceocnodnudcutecdtetdo tporperdeidcticEtRERbinbdinedrserosronronno-nbi-nbdinedrser[s42[4,423,4] 3a]nadnadgaognoisntisstosroarnatangtaognoisntisst[s44[4,445,4].5].

MMaannyylilgigaanndd-b-baasseeddccoommppuutatatitoionnaal lmmeeththooddsswweerereuusseeddttoopprreeddiicctt EERR aaccttiivviittyy ooff cchheemmiiccaallssbbaasseeddoonncchheemmiiccaallffeeaattuurreess,,iinncclluuddiinnggEERRbbiinnddeerrssaannddnnoonnbbiinnddeerrss,,aannddaaggoonniisstt and antagonist activities [31,46?49]. However, the dynamic binding patterns of ER agonists

and antagonists are not clearly understood. Hence, in this study we applied QM-Polarized

Ligand Docking (QPLD) and MD simulations to elucidate the dynamic binding patterns of ER agonists and antagonists using 17-estradiol (E2) and 4-hydroxytamoxifen (OHT).

Int. J. Mol. Sci. 2021, 22, 9371

and antagonist activities [31,46?49]. However, the dynamic binding patterns of ER ago

nists and antagonists are not clearly understood. Hence, in this study we applied QM

Polarized Ligand Docking (QPLD) and MD simulations to elucidate the dyn3aomf 1i8c binding patterns of ER agonists and antagonists using 17-estradiol (E2) and 4-hydroxytamoxi

fen (OHT). E2 is the natural steroid hormone that activates ER. The activated ER modu

Eelqxa2upteeirssensttghsleyieon,nntehaineteuxcdrpeailrlmlesss.etsEerir2oionnbidtieninrhdaosccrewtmlslisotwh.nEieEt2hRthbtiahnitnetadhecesstitnwvruoaicttgelheesunEEsRrRae.nisnTdphotfhoenresamecntseuivalcealdmteeiumdesneEart.RnoSdfumEbfoosRderqumaulnasedtneatrsldeygg,iemtunhleeeart.eSsutrbasne dsicmrieprtiionnteroafcttshwe ittahrtgheet egsetrnoeg.eEn2rehsapsontwseoelhemydernotxoyflEgRroanudpsre, gounleataest tCra3nascnrdipatinoontohfer at 17 t(hFeigtaurrgeet2g).enTeh.eE2hyhdasrotwxyolhgyrdoruoxpyal tgrCo3upfos,romnse ahtyCd3roagnednanbootnhdersawt 1it7h G(Fliug3u5re32a)n. TdhAe rg394 o hEyRd.roTxhyel g1r7ou-pOaHt Cg3rofourpmfsohrymdsroagehnybdornodgsenwibthonGdluw35i3thanHdisA5r2g43.9T4 hoef EpRla. nThare 1p7art-OoHf A/B ring

gfororumpsfoarsmasnadhwyidcrhogbeentwboenend wAiltah3H50isa52n4d. TLheeup38la7n.aTrhpearDt orfinAg/Bforrinmgsfoarnmosnapsoalnadrwcoicnhtact with bIleet4w2e4e,nGAlyla532510aanndd LLeeuu538275.[7T]h.eODHrTinigs afosremlescatinvoenepsotrloargeconnrteaccet pwtiothr mIleo4d2u4,laGtolyr5a2n1d acts a aanndaLnetua5g2o5n[i7s]t. OtoHwTarisdas sEeRlecitnivsepeestcriofigcentisrseuceepsto[6r ]m. Todhuelahtyordarondxyalctgsraosuapn ianntOagHoTnis(tFigure 2 ptahHofafiaw1rsnt2aiaortdayfhwsttiohEgaweRhyaAibnrtFdoisn2spodEseciiRccntieufig[,5cpb0aty]lifo.sfspicBnukaineiirtnsdytg[i6ontc]ogf.owtToahfhcaetOeridvhAHasyTFtdEo2rirRnossxti[byht5ielen0,g]dLr.bBioBnluPogipcnokidfinniEinnAORggFHpc2oTuof[sa(5hOFc1eit]Hgsi.vutTIhraneetion2QHr)stP1hh2LabesDainLwa, dBhtahiiyPgnethgooafbboiiEnncicRndAuiitnppFiogyu2s[h5e1s].thIn mQoPlLecDu,latrhcehaarbgeinsiwtioeremaoplpelciuedlatrocohbatarginesbiwndeirnegaopripelnietadtioton oofbtthaeintwboincdhienmgicoarliseinntathtieon of th LtwBPoocfhEeRmic[5a2ls].iMn tDhesiLmBuPlaotifoEnsRwe[r5e2u].sMedDtoseimlucuildaattieonthsewdeyrneamusicedbitnodeinlugcpidatateterntshoefdynami tbhienadgionngisptaEt2tearnnds aonfttahgeonaigstoOniHstTE. 2 and antagonist OHT.

FFiigguurere2.2T. wTwo-od-idmiemnseinosniaolnsatrluscttruurcetsuorfe1s7of-e1s7tra-deisotlra(Ed2i)oal n(Ed24)-haynddro4x-hytyadmrooxxiyfetnam(OoHxiTf)e.n (OHT).

2. Results

22..1.RMesoulelctuslar Docking

2.1. MQPoLleDcualnardDGolicdkeindgocking are two widely used docking methods to identify orientations oQfPcLomDpaonudndGsliidnebidnodciknginsgitaesreoftwprootweiindse[l5y2?u5s4e]d. TdhoeckEiXntgram-Perethciosidosnt(oXiPd)eGnltiidfye orienta sotscircooioernnresesta,sotQ,ifQoPcnLPosDLmaDrspecsooscrhueoonsrwdeasnnsdaiinnnddTobacdibknolidencgik1n.iegnngesriegtneysevroagflyupevsraooltufeteihnsesoffo[5tuh2r?ec5of4om]u.prTlechxoeemsEwpXlietthrxaet-hsPewrbeiectshist itlohigneanb(XdesPt)liGgalindd Toarbileen1t.aDtioocnkisnagrsecosrhesoawndndinockTianbgleene1r.gy values for the four ER complexes.

TaEbRlCeo1m. pDleoxcking scores and doGclkidine g energy values for the four ER cQoPmLDplexes.

XP Score Kcal/mol Docking Energy QPLD Score Kcal/mol Docking Energy

EREERRCo12m__EEp22 lex

-11.00 Glide -39.74 XP Sco-re9.6K5 cal/mol Dock-in32g.5E4 nergy

ER1_OHT

-9.07

-29.43

EERR2_1O_HET2

--181.5.900

--3359..8714

ER2_E2

-9.65

-32.54

-11.45

QPLD-38.78

QPLD-9S.8c2ore Kcal/mol -33D.41ocking Energy

-8.17

-28.96

-10.9-411.45

-38.23 -38.78

-9.82

-33.41

ERIn1b_OotHh Tdocking m-e9t.h0o7ds, the agoni-st29E.243had a lower dock-in8.g17energy in the agon-i2s8t.96 coEnRfor2m_aOtiHoTn (ER1) t-h8a.n59in the antago-n3is5t.8c1onformation (ER-120).,94while the antagon-i3s8t.23

OHT had a higher docking energy in the agonist conformation (ER1) than in the antagonist

conforInmabtoiothn (dEoRck2in).gThmeeothrioendtsa,titohnesaogfoEn2isatndE2OhHaTdina EloRwe1radndocEkRing2,eansewrgelyl ains tthhee agonis

EcRonfroersmidauteisoinnt(eEraRcti1n)g twhiathn Ein2 atnhde OaHntTaginotnhiestfocuorncfoomrmplaetxieosn, a(rEeRdep2i)c,tewdhinileFitghuerea3n.tagonis

EO2HinTthheabdinadhinigghsietre dofoEcRkin1gfoernmesrginyteirnacthtioenasgwointhisGt cluo3n5f3o,rAmrga3ti9o4n, a(nEdRHi1s)52t4h.aEn2ianlstohe antag foonrmisst icnotnerfaocrtmioantsiownith(EGRlu325).3Tahnde oArrige3n9t4atbiuotnfsaiolsf tEo2inatnedracOt HwTithinHEisR5241inanthdeEbRind2in, gas well a TsbtuihihtrneeeedoiEi3nfn.RtgEeERrs2airtciee2tnis.ooiOtfndhEHuaeRneTbasifl2noiy.nrsdOmtiisenHsrrgaeThvcysitendiiatntreleoegrodgawefctnhtEistabRhwtoEEnit12d2hffiaAoonnrrrtmmgedr3sasO4cm4itHniobotTruneetsrihfanwaycitditlhtsirhoeotnobgfsooeintnuwhtrebiArotcahnrocgdmtG3w4ilpnu4itlt3eeahr5xnaG3edc,sltuG,iAo3aln5rurg3se335iidnn39e4EiEpn,RRaitchnt11eed,.dHinis5F2ig4

E2 also forms interactions with Glu353 and Arg394 but fails to interact with His524 in the

binding site of ER2. OHT forms hydrogen bond interactions with both Arg344 and Int. J. MolG. Slcui. 2305213, 2i2n, 93t7h1e binding site of ER2. OHT interacts with Arg344 but fails to interact with4 of 18

Glu353 in ER1. The interaction analysis revealed that E2 forms more hydrogen bond

interactions in ER1, while OHT forms more hydrogen bond interactions in ER2. These four complexes wewrehisleuObHjeTctfeodrmtsomMorDe hsyimdrouglaentiboonnsdtionteelruacctiidonasteintEhRed2y. Tnhaemseicfobuirncdominpglepxeast-were terns of agonist E2saunbjdecatendtatogMonDisstimOuHlaTtioinns EtoRelu.cidate the dynamic binding patterns of agonist E2 and

antagonist OHT in ER.

FigFuirge u3.reBi3n.dBiningdoirniegntoartiioennstaoftiEo2nasnodf OEH2 TanindtOheHhTydirnopthheobhicydbirnodpinhgopboicckbeitnodf iEnRgp1oacnkdeEtRof2E. RThe1daontdtedERline2s. repTrehseendt hoytdterdogelinnbeosnrdeipnrteersaecntitonhsy. Gdrreoegnecnircbleosnrdepriensteenrtatchteiohnyds.roGphreoebnic rceisricdlueess;rceyparnecsiercnletstrheeprehsyedntrtohpehpooblairc resirdeuseids;ureeds;ancdyabnlueciorvcallessrerperperseenstetnhte tnheegaptiovelaarndrepsoidsiutivees;chraerdgeadnrdesibdluuees, orevsaplesctriveeplyr.esent the negative and

positive charged residues, respectively. 2.2. MD Simulations

2.2.

MD

Simulationds ockiInngt,hsetrMucDtusrimesuwlaetrieonrescfoorrdeeadchfoorfetvheerfyou4.r8cpoimcopsleecxosntdrusc(tpusr)esinotbhteaitnreadjecfrtoomry

molecular file. Thus,

In the MD simeuaclhattiroanjesctfooryr efialecchonotfatinhse2f0o,8u3r5csotrmucptulerexs.sTtrhuecdtuetraeilss obf tthaeinseimduflraotimonmsyostleemc-s are

ular docking, strucstuumremsawrizeerde irneTcaobrlde e2d. for every 4.8 picoseconds (ps) in the trajectory file.

Thus, each trajectoTrayblfeil2e. Scuomnmtaairny sof2t0he,8M3D5 ssitmruulcattiuonressys.tTemhse. details of the simulation systems

are summarized in Table 2.

Complex

Atoms in Complex

Waters

Ions

Table 2. Summary of the MEDRs1im_Eu2 lation systems.3980

Complex ER1_E2

ER1_OHT

3994

AERto2m_Es2 in Complex 3946

ER2_OHT3980

3960

ER1_OHT

3994

7890

7890

Waters 9138 7890 9138 7890

30 Na+; 23 Cl-

29 Na+; 22 Cl-

Io36nNsa+; 26 Cl- 30 Na+35; N23a+C; 2l5-Cl- 29 Na+; 22 Cl-

ER2_E2

To understa3n9d46the dynamics of ER bind9i1n3g8with the agonis3t 6E2Nanad+;a2n6taCgoln-ist OHT,

ER2_OHT root mean squa3re9d6e0viations (RMSD) were9c1a3lc8ulated between3t5heN20a,+8;3525stCrulc-tures for

ER1_E2, ER1_OHT, ER2_E2, and ER2_OHT using a MATLAB script. The obtained

RMSD matrixes for the four complexes are shown in Figure 4. Examining the RMSD values

To understandfrothmetdheynMaDmsiicmsuolfatEioRnsb(isnhdoiwnng iwn iFtihguthree4a)gfoounnidsttEha2tasntrducatunrtaalgcohnanisgteOs wHeTre, not root mean square tdheevsiaamtieodnusri(nRgMthSeDM)DwseimreulcaatilocnuslanteddthbeeRtwMSeDenmtahtreix2e0s,f8o3rm5 esdtrpuacttuerrness. Ffuorrther-

ER1_E2, ER1_OmHoTre, ,EthReR2M_ESD2,paanttdernEsRare2d_iOffeHreTntuamsinonggathMe fAouTrLcoAmBplsecxreisp, tin. dTihcaetiongbttaheinbeindding RMSD matrixes fordythnaemfiocus rofctohme apgloenxisetsaandreanshtaogwonnistininFEiRguarere4d.ifEfexraenmt.ining the RMSD val-

ues from the MD simulations (shown in Figure 4) found that structural changes were not

the same during the MD simulations and the RMSD matrixes formed patterns. Furthermore, the RMSD patterns are different among the four complexes, indicating the binding dynamics of the agonist and antagonist in ER are different.

IntI.nJt..MJ. oMl. oSl.ciS.c2i0. 210,2212, ,2923, 7x1FOR PEER REVIEW

5 o5f o18f 19

FigFuigreur4e. 4R.MRMSDSDmamtraitxreixsefsrofmromMDMDsimsiumlautliaotniosnosfothf ethfeouforucrocmopmlepxleexs:es(A: ()AE)RER1_1E_2E, 2(B, ()BE)RER1_1O_HOTH,T(C, ()CE)RER2_2O_HOTH, Tan, adnd (D()DE)RER2_E2_2E. T2.hTehceomcopmlepxleisxsihsoswhonwinntihnetthiteletiotlfeeoafcheapcahnpela.nTehl.eTtihmeetiomf ea ostfraucsttururectiunrtehienMthDe sMimDuslaimtiounlastisondsepisicdteedpibcyted thebyaxtihse. Taxhies.RTMheSDRMvaSlDuevsaalrueecsoalroer coldoerdcoads esdhoaws nshionwthneinsutbhfiegsuurbeficgoulorer lceogloenr dlesg. ends.

ThTehienitnertearcaticotinoneneenrgerygwy awsacsalccaulclautleadteudsuinsginpgripmriemMe MM/MG/BGSBASAfofrowr hwohleolteratjreacjetocrtoyry filfeisleasnadndthtehceaclcaulcluatleadteedneenrgerygvyavluaeluseasrearperpovroidveiddeidn iTnaTblaeb3le. 3.

TaTbalebl3e. I3n. tIenrtaecrtaicotnioenneenrgeyrgvyavluaelus efosrfothrethEeREcRomcopmlepxleesx.es.

AAccrroonnyymms s GG_B_Binindd

EERR11__EE22 --444.41.100

EERR11__OOHHTT EERR22__EE22 ER2_OHT

ER2_OHT

--444.46.688 --242.46.699 --424.22.244

G_BBiinndd__CCoouulolmomb b

--77..7733 --2222..0033 --99..1199 --3399..6655

G_GBi_nBdi_nvdd_WvdWLigLaingdaEnndeErgnyergyCoCmopmlexplEenxeErgnyergyReRcepcetoprtEonr eErngyergy

-22-.2172.17

1.731.73

-94-0924.7012.71

-9-39603.6304.34

-17-.1477.47 -7.-137.13 -17-.1047.04

32.4332.43 1.721.72

32.12

32.12

-93-5923.9572.97 -93-2953.7295.79 -93-4923.1432.13

-9-39403.4701.71 -9-39023.0821.81 -9-39323.3021.01

To identify distinct structural patterns, hierarchical clustering analysis was con-

ducTtoedidbeansteifdy odnisttihnectRsMtruSDctumraaltrpiaxtetse.rnTsh,ehmieraajorcrhcilcuasltcelruss(tewriinthg>an5a0l0yssitsruwctausrceosn) dfruocmtedthe baMseDd osinmthuelaRtiMonSsDfomraEtrRixe1s_.ET2h,eEmRaj1o_rOclHusTte, rEsR(w2it_hE>2,5a00ndstrEuRctu2r_eOs)HfrTomartehseuMmDmsairmizuelda-in tioTnasbfloer4E. R1_E2, ER1_OHT, ER2_E2, and ER2_OHT are summarized in Table 4.

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