Deciphering Modern Glucocorticoid Cross-pharmacology Using Ancestral Corticosteroid Receptors

12 Pages·2012·2.52 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.287,NO.20,pp.16267–16275,May11,2012
©2012byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
Deciphering Modern Glucocorticoid Cross-pharmacology
Using Ancestral Corticosteroid Receptors□S
Receivedforpublication,January25,2012,andinrevisedform,February21,2012Published,JBCPapersinPress,March21,2012,DOI10.1074/jbc.M112.346411
JeffreyA.Kohn,KirtiDeshpande,andEricA.Ortlund1
FromtheDepartmentofBiochemistryandtheDiscoveryandDevelopmentalTherapeuticsProgram,WinshipCancerInstitute,
EmoryUniversitySchoolofMedicine,Atlanta,Georgia30322
Background:Drugsthattargetsteroidreceptorsarenotoriouslypromiscuous,causinganarrayofoff-targetsideeffects.
Results:ReversalofthehistoricalmutationH853Rinthemineralocorticoidreceptor(MR)fullyrestoresagonistactivityby
mometasonefuroate,anMRantagonist.
Conclusion: A single residue outside of the ligand-binding pocket toggles agonism versus antagonism response by MR to
syntheticligands.
Significance:Ancestralproteinsareidealtoolstoelucidatethemechanismsofdrugselectivity.
Steroid receptors (SRs) are the largest family of metazoan reproductivediseases,andcancer.Overtime,avastrepertoire
transcription factors and control genes involved in develop- ofreceptorshasevolvedtorespondtosmallchemicalstimuli,
ment,endocrinesignaling,reproduction,immunity,andcancer. making them attractive pharmacological targets. However,
The entire hormone receptor system is driven by a molecular becausemostreceptorsbelongtolargeclassesofevolutionary-
switchtriggeredbythebindingofsmalllipophilicligands.This relatedproteinsthatshowhighstructuralsimilarity,targetinga
makes the SRs ideal pharmaceutical targets, yet even the best single receptor subtype is a major challenge. Poor selectivity
clinically approved synthetic steroidal agonists are prone to can cause serious off-target side effects, as seen in the treat-
cross-reactivity and off-target pharmacology. The mechanism mentsofmajordepression(3),heartdisease(4,5),asthma(4,5),
underlying this promiscuity is derived from the fact that SRs andallergies(6).
sharecommonstructuralfeaturesderivedfromtheirevolution- To fully understand the mechanisms supporting receptor-
aryrelationship.Moreoftenthannot,rationalattemptstoprobe ligandrecognition,selectivity,andactivation,robuststructure-
SRdrugselectivityviamutagenesisfailevenwhenhighquality functionrelationshipsmustbebuiltfromextensivemutational
structuralandfunctionaldataareavailableduetothefactthat analysisandliganddesign.Thisanalysisishinderedforseveral
important mutations often result in nonfunctional receptors. reasons.First,aminoacidresiduesconferringproteinfunction
ThishighlightsthefactthatSRssufferfrominstability,prevent- and ligand specificity between homologous receptors can be
ingin-depthmutationalanalysisandhamperingcrystallization difficult to identify among the vastly more prevalent neutral
of key receptor-ligand complexes. We have taken a unique mutations that accumulate over time (7). Second, restrictive
approachtoaddressthisproblembyusingaresurrectedances- mutations that are not directly related to the protein-ligand
tralproteintodeterminethestructureofapreviouslyintracta- interactioncanaccumulateinextantproteins,preventingthe
blecomplexandidentifiedthestructuralmechanismsthatcon- tolerance of function-shifting mutations (8). Third, many
fer activation and selectivity for a widely used glucocorticoid, mutationsaredestabilizingandresultinlossofproteinfunc-
mometasonefuroate.Moreover,wehaveidentifiedasingleres- tion,complicatingthedistinctionbetweenaneffectthatisspe-
iduelocatedoutsideoftheligand-bindingpocketthatcontrols cific to the protein-ligand interaction versus an effect that is
mometasonefuroateantagonismversusagonisminthehuman globallyinactivatingtotheprotein(7).Althoughmostconclu-
mineralocorticoidreceptor. sionsarecurrentlydrawnfromfunction-killingmutations,the
insightneededtounderstandligandselectivityamongaclassof
homologous proteins would be better drawn from function-
Complexlifedependsonintra-andintercellularcommuni- shiftingmutationsthatpreservereceptoractivation.
cation, whereby secreted messengers are detected by specific Theseproblemshavehinderedthedesignofselectivedrugs
receptorstoregulatemetabolism,reproduction,cellcycles,and thattargethumansteroidreceptors(SRs).2SRsareafamilyof
more.Thiscoordinationtightlycontrolscellularactivitywithin ligand-regulated transcription factors that control genes
thehigherorganism.Poorcoordinationoftheseprocessescan involved in development, endocrine signaling, reproduction,
resultinmanyhealthconcerns,includingmetabolicdisorders, immunity,andcancer(9).Thismakesthemattractivepharma-
ceutical targets. Although SRs show exquisite selectivity for
□S ThisarticlecontainssupplementalFigs.S1andS2.
Theatomiccoordinatesandstructurefactors(code4E2J)havebeendepositedin 2Theabbreviationsusedare:SR,steroidreceptor;PR,progesteronereceptor;
theProteinDataBank,ResearchCollaboratoryforStructuralBioinformatics, AR,androgenreceptor;MR,mineralocorticoidreceptor;GR,glucocorticoid
RutgersUniversity,NewBrunswick,NJ(http://www.rcsb.org/). receptor; AncSR, ancestral SR; AncGR, ancestral GR; TIF2, transcription
1To whom correspondence should be addressed: Dept. of Biochemistry, intermediaryfactor2;MOF,mometasonefuroate;ANOVA,analysisofvari-
EmoryUniversitySchoolofMedicine,Atlanta,GA30322.Tel.:404-727- ance;HSD,honestlysignificantdifference;AncCR,ancestralcorticosteroid
5014;Fax:[phone];E-mail:[email protected]. receptor;LBP,ligand-bindingpocket.
MAY11,2012•VOLUME287•NUMBER20 JOURNALOFBIOLOGICALCHEMISTRY 16267
This is an Open Access article under the CC BY license.
DecipheringGlucocorticoidCross-pharmacologyUsingAncSRs
theirendogenoushormones,SR-targetingdrugstendtobepro- transformed into Escherichia coli strain BL21(DE3) and
miscuousandcausemanyoff-targetsideeffects(10,11).Thisis expressed as a maltose-binding protein-His fusion. Cultures
becauseSRs,consistingoftheestrogenreceptor,progesterone (1.3litersinTerrificBroth)weregrowntoanA of0.6–0.7
600
receptor (PR), androgen receptor (AR), mineralocorticoid andinducedwith400(cid:1)Misopropyl(cid:2)-D-thiogalactopyranoside
receptor (MR), and glucocorticoid receptor (GR), descended and 50 (cid:1)M MOF at 30°C for 4 h. Cell mass was collected by
fromacommonancestor(cid:1)500millionyearsago(seeFig.1A) centrifugation at 4000 (cid:2) g for 15 min, lysed, and purified by
and show high structural similarity (9, 12). In the absence of nickelaffinitychromatography.Themaltose-bindingprotein-
ligand, SRs remain partially unfolded and associate with heat Histagwascleavedbytobaccoetchvirusproteaseat4°Cover-
shockproteins(13,14).Thisinstabilityisnecessarytopermit nightwithsimultaneousdialysisintobuffercontaining300mM
theconformationalchangesthatdrivereceptoractivationupon NaCl, 20 mM Tris (pH 7.4), 5% glycerol and purified to
ligandbinding(1,2,7,14–16),butithaslimitedourabilityto homogeneity by nickel affinity, followed by gel filtration
probe receptor-ligand interactions via mutagenesis, as many chromatography.
mutationsofinterestdisabletheproteinentirely. Crystallization, Data Collection, and Structural Refine-
Withtheadvancementofwhole-genesynthesisandpioneer- ment—PureAncGR2wasconcentratedto3–5mg/mlinbuffer
ingeffortsmadeincomputationalandevolutionarybiology,it containing300mMNaCl,20mMTris(pH7.4),5%glycerol,50
is now possible to predict and “resurrect” ancestral genes. (cid:1)MCHAPS,and50(cid:1)MMOF.Crystalsweregrownviahanging
Ancestral gene reconstruction is used to study the molecular dropvapordiffusionat4°Cfromsolutionscontaining0.75(cid:1)lof
evolutionofabiologicalsystem(12,17–19)butshowspromis- AncGR2-TIF2-MOF solution, 0.75 (cid:1)l of 1.5–3 M ammonium
ing applications to the process of drug design. By comparing formate,andadodecapeptidederivedfromtheGRcoactivator
twoancestralproteinsfromnodesonanevolutionarytree,we human TIF2 ((cid:3)H N-ENALLRYLLDKD-CO(cid:4), SynBioSci
3 2
are provided with a smaller subset of possible amino acid Corp.).Crystalswerecryoprotectedbyimmersionin1.5–3M
replacementstodissectbetweenrelatedproteinsthathavedif- ammoniumformatecontaining25%glycerolandflash-frozen
ferentligandspecificities.Oureffortscanbefocusedonfewer inliquidnitrogen.Datatoaresolutionof2.5Åwerecollectedat
residues when probing structure-function relationships than the Southeast Regional Collaborative Access Team at the
whenlookingonlyatextantproteins.Thisapproachtherefore AdvancedPhotonSource(Argonne,IL)(Table1).Thestruc-
allows us to avoid interference from neutral and restrictive tureoftheAncGR2-MOF-TIF2complexwassolvedbymolec-
mutations that have accumulated over time. Furthermore, ular replacement using PHASER in the CCP4 software suite.
unlikemanyextantproteins,ancestralproteinsshowremarka- ModelbuildingandrefinementwereperformedusingRefmac
ble tolerance toward changes in function-shifting residues, and Coot. Cavity volumes were calculated using CASTp, and
makingthemmorestableunderlaboratoryconditions. figuresweregeneratedinPyMOL.TherefinedAncGR2-MOF
Wehypothesizedthatonecouldexploittheseancestralpro- structure has been deposited in the Protein Data Bank (code
teinstounderstandcross-pharmacologyinhumanSRs.Ances- 4E2J).
tral SRs (AncSRs) are more tolerant to mutation than their Mutagenesis—Wild-type AncGR1 (GenBankTM accession
extant descendants (20), and their molecular and structural number JF896321.1) and AncGR2 were subcloned into a
evolutionhasalreadybeencharacterized(8,12,20–23).Anc- pMCSG7-maltose-bindingprotein-Hisexpressionvector,and
SRsthereforemakeaneffectivemodeltostudythestructural the following mutations were created from these constructs:
mechanisms of SR pharmacology. To achieve this goal, we AncGR1-S106P, AncGR1-S106P/L111Q, AncGR1-S106P/L111A,
determinedthestructureoftheancestralglucocorticoidrecep- AncGR2-P106S, AncGR2-P106S/Q111L, and AncGR2-P106S/
tor2(AncGR2)ligand-bindingdomainincomplexwithafrag- Q111A.MutagenesiswasperformedusingaQuikChangeIIXL
mentofhumantranscriptionintermediaryfactor2(TIF2)and kit(Stratagene).
mometasone furoate (MOF). We draw upon functionally Ligand Binding Assays—Wild-type or mutant AncGR1 or
importanthistoricalaminoacidsubstitutionstoelucidatethe AncGR2wasexpressedasdescribedaboveandassayedpriorto
mechanismsdrivingGRactivationforthiswidelyusedgluco- tobaccoetchvirusproteasecleavageaspurifiedmaltose-bind-
corticoid. Furthermore, we use a combination of structural ing protein fusion proteins. All fluorescence polarization
analysisandfunctionalassaystoexplaintheselectivityofthis experiments were performed in buffer containing 150 mM
drugagainstMRandARandstrongcross-reactivitywithPR. NaCl, 10 mM HEPES (pH 7.4), 5 mM DTT, 3 mM EDTA, and
0.005%Tween20.Bindingaffinityfordexamethasone-fluores-
EXPERIMENTALPROCEDURES
ceinwasmeasuredwithaconstantconcentrationof12nMdex-
Chemicals and Reagents—Chemicals were purchased from amethasone and a variable protein concentration of 10(cid:4)10–
SigmaorFisher.ThepMALCH10TandpMCSG7-basedpro- 10(cid:4)5 M. Competition assays were performed at a protein
teinexpressionvectorsweregiftsfromJ.Tesmer(Universityof concentration1.2timesitsbindingaffinityfordexamethasone
Texas, Austin) and J. Sondek (University of North Carolina, in the presence of 12 nM dexamethasone and 10(cid:4)10–10(cid:4)5 M
ChapelHill),respectively.TheemptypSG5-basedmammalian competingligand.DatawereprocessedwithGraphPadPrism5.
expression vector and human MR and GR pSG5 constructs, Statisticalsignificancewasdeterminedbytwo-factoranalysisof
pFRlucreporter,andphRLtkreporterweregiftsfromJ.Thorn- variance(ANOVA),andindividualcomparisonsweremadewith
ton(UniversityofOregon,Eugene). Tukey’shonestlysignificantdifference(HSD)posthoctests.
ProteinExpressionandPurification—AncGR2(GenBankTM In-cellActivationAssays—Allancestral,andmutantligand-
accessionnumberEF631976.1)inapMALCH10Tvectorwas binding domains were cloned into a pSG5 expression vector
16268 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER20•MAY11,2012
DecipheringGlucocorticoidCross-pharmacologyUsingAncSRs
TABLE1
Datacollectionandrefinementstatistics
AU,asymmetricunit;r.m.s.d.,rootmeansquaredeviation;PDB,ProteinDataBank.
Resolution(highestshell;Å) 2.50(2.59–2.50)
Spacegroup P6
Unitcelldimensions a(cid:5)1104.4,b(cid:5)104.4,c(cid:5)143.9Å;(cid:3)(cid:5)(cid:2)(cid:5)90.0°,(cid:4)(cid:5)120.0°
No.ofreflections 30,710
R (highestshell)a 7.7%(42.2%)
sym
Completeness(highestshell) 99.90%(98.96%)
Averageredundancy(highestshell) 8.0(7.9)
I/(cid:5) 29.3(5.3)
Monomers/AU 2
No.ofproteinatoms/AU 4195
No.ofligandatoms/AU 85
No.ofwaters/AU 151
R b(R )c 20.5(25.5)
working free
AverageB-factors(Å2)
Protein 45.0
Ligand 53.5
Water 45.5
r.m.s.d.
Bondlengths(Å) 0.005
Bondangles 1.078°
PDBcode 4E2J
a R (cid:5)(cid:6)(cid:1)I(cid:4)(cid:7)I(cid:8)(cid:1)/(cid:6)(cid:1)I(cid:1),whereIistheobservedintensity,and(cid:7)I(cid:8)istheaverageintensityofseveralsymmetry-relatedobservations.
bRsym (cid:5)(cid:6)(cid:2)F(cid:1)(cid:4)(cid:1)F(cid:2)/(cid:6)(cid:1)F,whereF andF aretheobservedandcalculatedstructurefactors,respectively.
cRwork(cid:5)ing(cid:6)(cid:2)F(cid:1)(cid:4)o(cid:1)F(cid:2)/(cid:6)c(cid:1)F foro7%oftheodatancotusedatanystageofthestructuralrefinement.
free o c o
immediatelyfollowingaGal4DNA-bindingdomainandaGR
hingesequence.CHO-K1cellsweregrownandmaintainedin
phenolred-freecomplete(cid:3)-minimalessentialmedium(Invit-
rogen)supplementedwith10%charcoal/dextran-strippedFBS
(Invitrogen) and penicillin/streptomycin. Cells grown in
96-well assay plates were transfected at 70–90% confluence
with1ngofreceptor,100ngofupstreamactivatorsequence-
drivenfireflyluciferasereporter(pFRluc),and0.1ngofconsti-
tutiveRenillaluciferasereporter(phRLtk)for4husingLipo-
fectamine 2000 in Opti-MEM I (Invitrogen). Transfections
wereendedbyreplacementwithcomplete(cid:3)-minimalessential
medium, and cells were allowed to recover overnight. After
recovery, cells were treated in triplicate with 10(cid:4)12–10(cid:4)6 M
ligand or vehicle (Me SO) in complete (cid:3)-minimal essential
2
mediumfor24h(finalworkingMe SOof1%)andthenassayed
2
with Dual-Glo luciferase substrate (Promega). Firefly activity
was normalized to Renilla activity, and the -fold increase in
activationwascalculatedrelativetothevehiclecontrol.Dose-
responsecurvesweregeneratedinGraphPadPrism5.Statisti-
cal significance was determined by two-factor ANOVA, and
individualcomparisonsweremadewithTukey’sHSDposthoc
tests.
RESULTS
FIGURE1.Evolutionaryhistoryofcorticosteroidreceptorsandstructure
AncGR2-TIF2-MOF Crystal Structure—MOF is a powerful ofAncGR2-TIF2-MOF.A,simplifiedphylogenetictreedepictingtheevolu-
topicalanti-inflammatorydrugfortheskinandairwaysandis tionofcorticosteroidreceptors.Activatinghormonesarelistedontheright.
ER,estrogenreceptor;ERR,estrogenreceptor-relatedreceptor.B,structureof
the active ingredient of Nasonex, Asmanex, and Elocon (24).
AncGR2(red)incomplexwithhumanTIF2(green)andMOF(cyan).C,AncGR2
Although MOF has been in clinical use for over 24 years, it LBPresidues(red)withMOFshown(cyan).
suffersfromseverecross-pharmacology,resultinginunwanted
side effects and limiting its use to topical applications. MOF bindingrequiresarearrangementofthehelixH6-H7regionof
strongly activates GR, cross-reacts with PR, and is selective the receptor to accommodate the large 17(cid:3)-furoate moiety,
against AR and MR. The ternary AncGR2-TIF2-MOF crystal inducing a 200-Å3 (1.3-fold) increase in the volume of the
structurerevealsthestructuralbasisforMOFbindingtoverte- ligand-binding pocket relative to dexamethasone; this high-
brate GRs (Fig. 1, B and C, and supplemental Fig. S1). The lightstheabilityofSRstoexpandtheirligand-bindingpockets
hydrogenbondnetworkthatisrequiredforactivationofcorti- to accommodate exogenous ligands (26, 27). The AncGR2-
coidreceptors(25)isintactandisstabilizedbyadipole-dipole TIF2-MOFstructurealsorevealsthatastrongH-bondisnot
interactionbetweenMOFC21-ClandAncGR2Asn-33.MOF possiblebetweenMOFandGln-111ofGR(Fig.1C),aninter-
MAY11,2012•VOLUME287•NUMBER20 JOURNALOFBIOLOGICALCHEMISTRY 16269
DecipheringGlucocorticoidCross-pharmacologyUsingAncSRs
Structural and Evolutionary Basis for Selectivity against
MR—Ourstructurealso suggestsamechanismfortheselec-
tivityofMOFagainstMRandAR.H6andH7,whichborderthe
17(cid:3)-bindingarea,arepartiallyunwoundandstabilizedbyPro-
637/Pro-106inGR/AncGR2,accommodatingthefuroatemoi-
ety(Fig.3A,left);MR,AR,andAncGR1haveaserineatthissite
that caps H7, positioning the helix within 2.5 Å of where the
furoate would rest, creating a steric incompatibility (Fig. 3A,
right).Wehaveshowninpreviousworkthat,duringtheevolu-
tionofGR,S106PandL111Qsubstitutionswerecriticalinboth
reshapingtheH6-H7regionofthereceptorandgeneratinga
new H-bond with the 17-OH moiety of cortisol, the endoge-
nousglucocorticoid(8,22).Totesttheeffectofreversingthese
criticalsubstitutionswithrespecttoMOFbindingaffinity,we
generatedtwoAncGR2mutants,P106SandP106S/Q111L,and
measuredtheirbindingaffinitiesforcortisol,dexamethasone,
and MOF using fluorescence polarization competition assays
against dexamethasone-fluorescein. The P106S reversal
reducedtheaffinityofAncGR2forallthreeligandsbyonlyan
orderofmagnitude(Fig.3B).Thisresultwassurprisingbecause
theP106Smutationwasidentifiedasthedrivingforcebehind
theH6-H7rearrangementrequiredtoopenspaceintherecep-
torforspecificrecognitionofhormoneswithC17substituents
(13, 22). Because MOF binding requires this structural rear-
rangement(Fig.3A),Pro-106likelyplaysaroleinstabilizingthe
H6-H7loopinaproductivebindingmodebutisnotabsolutely
required to induce this structural change. AncGR2-P106S/
Q111L, which is known to be inactive to endogenous ligands
(8), did not bind dexamethasone-fluorescein (supplemental
Fig.S2).Thispreventedcompetitionassaysonthismutantbut
FIGURE2.Structuralbasisforoff-targetactivationofPR.A,thehumanPR
(hPR)-MOFcomplex(white;ProteinDataBankcode1SR7)superimposedon suggestedthatH7indeedrepositionstoplaceLeu-111incon-
theAncGR2-MOFcomplex(red).PRresiduePhe-794maintainsspaceforthe tactwithC17ofthesteroid.Thisgeneratesapolarincompati-
17(cid:3)-furoatemoietyandcontributesahydrophobicinteractionviathearom-
bility with C17-OH-containing steroids, such as cortisol and
atizedsidechain(15).PRresidues791ESSF794onH7appeartoplayakeyrolein
allowingstrongMOFbindingbypositioningtheH6-H7loopandH7viaa dexamethasone, and introduces a steric clash with the bulky
conservedGlu-791–Ser-793H-bond.B,thismotifisstrictlyconservedamong furoatesubstituentofMOF.Totestthishypothesis,wegener-
extantPRsbutisnotpresentinAncSR2.
ated a P106S/L111A mutant, designed to alleviate this steric
clash in the Pro-106 background, which restored binding to
action that plays a critical role in the specific recognition of MOFanddexamethasone(Fig.3B).Asexpected,cortisolbind-
17(cid:3)-OH-substitutedligandsandisabsolutelyrequiredforcor- ingwasonlymarginallyrestoredbecausecortisoldoesnotcon-
tisol activation (8). Instead, hydrophobic interactions replace taintheadditionalbulkyhydrophobicgrouppresentonMOF
thisinteractioninafashionanalogoustothestructureofthe tostabilizethecoreofthereceptorintheabsenceofthecritical
GR-fluticasonefuroatecomplex(28). Gln-111–17-OHH-bond.Interestingly,dexamethasonebind-
Structural and Evolutionary Basis for PR Cross-reactivity— ingwasmorefullyrestoredthancortisolbinding,presumably
TheAncGR2-TIF2-MOFstructureallowsforthedirectstructural duetoadditionalinteractionsonitsmodifiedbackbone.Thus,
comparison of GR-MOF and PR-MOF complexes and reveals theH6-H7regionofthereceptorcanadoptanexpandedcon-
how additional space in the H6-H7 loop region is created to formationintheabsenceofPro-106,suggestingthattheH6-H7
accommodatethefuroatemoiety.PRresidues791ESSF794onH7 region of GRs is inherently flexible, allowing it to adapt to
appear to play a key role in allowing strong MOF binding by ligand-inducedperturbation.Thisreshapesourunderstanding
expandingthepocketviaaconservedGlu-791–Ser-793H-bond of the role of the H6-H7 region within the ligand-binding
betweentheH6-H7loopandH7(Fig.2A).Stericbulkprovidedby domainintherecognitionofsyntheticglucocorticoids.
PRPhe-794betweenH7andH3maintainsspaceforthe17(cid:3)- WehaveshownpreviouslythatAncGR1,whichprecededthe
furoate moiety and contributes a hydrophobic interaction evolutionofAncGR2,isalowsensitivityMR-likereceptorwith
viathearomatizedsidechain(26).Thismotifisstrictlycon- activationbybothmineralocorticoidsandglucocorticoids(22).
served among PRs but is not present in AncSR2 (the com- BecauseMOFisselectiveagainstMR,wereasonedthatMOF
mon ancestor of all 3-keto-SRs) or AncSR3 (the common woulddisplaysimilarselectivityagainstAncGR1.Surprisingly,
ancestor of PR and AR) (Figs. 1A and 2B). Therefore, PR MOFboundAncGR1withanaffinitycomparablewithdexam-
responsetoMOFwasprobablyalateevolutionaryderivation ethasoneandcortisol(Fig.3C),indicatingthatAncGR1H7had
resultinginthiscross-reactivity. already acquired the plasticity needed to accommodate the
16270 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER20•MAY11,2012
DecipheringGlucocorticoidCross-pharmacologyUsingAncSRs
FIGURE3.BindingofMOFbymodernandancestralSRsrequiresexpansionoftheLBP.A,structureofthe17(cid:3)-bindingpocket.LikePRandAncGR2,human
GR(hGR)hasanextendedH6-H7loopconformation(left);MR,AR,andAncGR1haveatightenedH6-H7thatwouldcreateastericincompatibilitywiththe
furoate(right).BandC,thebindingaffinitiesofAncGR2(B)andAncGR1(C)mutantsfortheindicatedligandweremeasuredbyfluorescencepolarization
competitionwithdexamethasone-fluorescein(Dex).AncGR2-P106S/Q111L(AncGR2-SL)didnotbinddexamethasone-fluorescein,andcompetitionexperi-
mentscouldnotbeperformedforthisreceptor.Statisticalanalyseswereperformedusingtwo-factorANOVA,withTukey’sHSDposthoctestsusedfor
individualcomparisons.Comparisonsfoundtobestatisticallysignificanttop(cid:9)0.05aremarked.*,comparedwiththesameligandbindingforthewild-type
receptor;#,comparedwithdexamethasonebindingforthesamemutant;†,comparedwithMOFbindingforthesamemutant.AncGR1-SA,AncGR2-P106S/
Q111A;AncGR1-PQ,AncGR1-S106P/L111Q;AncGR1-PA,AncGR1-S106P/L111A.DandE,receptoractivationforGR-like(D)andMR-like(E)receptorswas
measuredbyDual-LuciferasereportergeneactivationintransientlytransfectedCHO-K1cellcultures.Themean(cid:10)S.E.isshown(n(cid:5)3).Statisticalanalyseswere
performedusingtwo-factorANOVA,withTukey’sHSDposthoctestsusedforindividualcomparisons.Comparisonsfoundtobestatisticallysignificanttop(cid:9)
0.05aremarked.*,comparedwithactivationofthesamereceptorbycortisol;#,comparisonsmadeasindicatedonthefigure.X,nobindingoractivation
observed.
bulkyfuroatemoiety.TheforwardmutationsAncGR1-S106P A Single Residue Controls MOF Selectivity and Transcrip-
andAncGR1-S106P/L111Qhadnoeffectsspecifictoapartic- tional Activity—To determine the structural differences
ular ligand, but the AncGR1-S106P/L111A mutation selec- betweenGRandMRthatgovernMOFrecognition,wecharac-
tively reduced cortisol binding while leaving dexamethasone terizedtheabilityofMOFtodriveluciferasereportergeneacti-
and MOF binding unaffected. This is presumably due to the vation across the entire ancestral corticosteroid phylogeny.
removalofa17(cid:3)-interaction.Thesedatashowthatreceptor- Although MOF only very weakly activated MR (Fig. 4A), it
ligand interactions at the 17(cid:3)-site are important for effective strongly activated AncGR1 and the ancestral corticosteroid
ligand binding, although poor interactions here can be sur- receptor (AncCR) with a subnanomolar potency, comparable
mounted by stronger interactions elsewhere along the ligand withthestrongactivationseeninAncGR2andGR(Fig.4,Aand
scaffold. B).Thisprovidesfurtherevidencethatthecorticoidreceptors
MAY11,2012•VOLUME287•NUMBER20 JOURNALOFBIOLOGICALCHEMISTRY 16271
DecipheringGlucocorticoidCross-pharmacologyUsingAncSRs
is unable to stabilize an active receptor conformation (25).
Thus,MRmusthaveaccumulatedepistaticchangesthatpro-
hibit activation from this drug. We therefore examined the
importanceofresiduesthatchangedonthelineageleadingto
MRwithrespecttoMOFactivation.Mutationofresiduesinor
near the ligand-binding pocket had no significant impact on
MOF activation without affecting receptor activation toward
cortisolanddexamethasone,consistentwithourfluorescence
polarization competition assays (Fig. 3, D and E) (data not
shown).Wethereforelookedforchangesoutsideoftheligand-
binding pocket and activation function surface. Arg-116 and
Gln-120inAncCR,correspondingtoHis-853andLeu-857in
MR,respectively,arelocatedonthesolvent-exposedfaceofH7
andinteractwiththemainchainoftheloopbetweenH5and
(cid:2)1, at AncCR residues Gly-87 and Met-89 (MR residues Ser-
824andPhe-826)(Fig.5A).Theseresiduesare(cid:11)14–18Åfrom
the furoate moiety of MOF and closest to the B ring of the
steroid(10–16Å),yetreversalofallfourresiduesinMRtotheir
ancestralstates(MR-GMRQ)completelyrestoredMOFactiva-
tion(Fig.5B).Wenarroweddownthecauseofthiseffect,first
tothoseresiduesonhelix7(hMR-RQ),andfurthertothesingle
residueatMRsite853.Reversalofthissiteviathesubstitution
H853R conferred full MOF activation (Fig. 5B). In wild-type
MR,His-853interactswiththemainchainatomsintheH5-(cid:2)1
loopandappearstostabilizetheMR-likeconfigurationofH7,
whichmustunwindtosupportactivationbyligandswithbulky
C17(cid:3)substituents. The stronger interaction provided by an
argininesubstitutionatthissitestabilizedMR-H853Rtoenable
MOFactivation(Figs.5Aand6).Importantly,thesechangesare
neutralwithrespecttoactivationbycortisol:neithertheEC
50
nor activation of cortisol was affected by the MOF selectivity
mutations(Fig.5,BandC),indicatingthatthestructuraldeter-
minantsofMOFactivationareuniquefromthosethatsupport
theendogenousligandrecognition.Introducingtheequivalent
forwardsubstitutionsinAncCR(AncCR-SFHL)failedtoabro-
gate MOF response (Fig. 5B), which is in line with the more
promiscuousphenotypeoftheancestralprotein.Intriguingly,
makingtheequivalentsitemutationshorizontallybetweenMR
and GR (MR-H853L/L857S and GR-L647H/S651L) not only
failedtoenableMOFactivationinMRbutalsoabrogatedacti-
vationbycortisolanddexamethasoneinGR(Fig.5,DandE).
MutationsattheseresiduesduringtheevolutionofGRswere
previouslyidentifiedtobedestabilizingtoGRs,contributingto
FIGURE4.Activationofmodernandancestralcorticosteroidreceptorsby
thelowaffinitybuthighselectivityofmodernGRsforendoge-
syntheticglucocorticoids.A,corticoidreceptorphylogenywithresponseto
syntheticglucocorticoids(dexamethasone(Dex)/MOF)isshown.Fullago- nous glucocorticoids (20). Here, disruption of this site in GR
nismisshowningreen,andweakornoagonismisshowninred.HumanGR fullydestabilizedtheactivereceptorduringcortisolanddexa-
(hGR)andMR(hMR)wereusedtorepresentextantmammalianGRandMR.
methasone binding. In contrast, MOF expanded the ligand-
The-foldactivation(B)andpotency(C)ofcorticosteroidreceptorligand-
bindingdomainsweremeasuredviaDual-Luciferasereportergeneactiva- bindingpocket(LBP)tomakeadditionalhydrophobiccontacts
tionintransientlytransfectedCHO-K1cells.Themean(cid:10)S.E.isshown(n(cid:5)3).
offeredbythefuroatering(Fig.6)andwasabletostabilizethe
Forthepurposeofthisresearch,activationbelow10-foldoverthecontrol(B,
red line) was considered weak agonism/antagonism, whereas activation active conformation, albeit at a much lower potency than in
abovethisthresholdwasconsideredfullagonism. wild-typeGR(Fig.5,DandE).
Weanticipatethatthefindingsproducedbythisstudywillbe
fromAncCRtoAncGR2areabletounwindH6-H7toaccom- applicabletoligandsthatprotrudeintoextrasteroidalbinding
modate the MOF 17(cid:3)-furoate moiety without requiring the regions within the LBP. Furthermore, the finding that ligand
S106Psubstitution. specificityisstronglyinfluencedbystructuralfeaturesthatlie
Furthermore,wehaveshownthatMOFexhibitsitsselectiv- welloutsideoftheLBPmustbetakenintoconsiderationduring
ityforGRoverMRnotviaadifferenceinpotencybutratherin thedevelopmentoffuturedrugs.Thefactthatthesesitescould
efficacy:althoughMOFbindsMR,withan(cid:11)100nMpotency,it notbeidentifiedusingextantproteinshighlightsthepowerof
16272 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER20•MAY11,2012
DecipheringGlucocorticoidCross-pharmacologyUsingAncSRs
FIGURE5.Distalresiduescontrolcorticosteroidspecificity.A,keyresiduespreceding(cid:2)-sheet1andH7inAncCRandhumanMR(hMR)werecross-mutated.
The-foldactivation(B)andpotency(C)weremeasuredviaDual-LuciferasereportergeneactivationintransientlytransfectedCHO-K1cells.Dex,dexametha-
sone.ThesameresiduesinGRandMRwerecross-mutated,andthe-foldactivation(D)andpotency(E)weremeasuredviaDual-Luciferasereportergene
activationintransientlytransfectedCHO-K1cells.Themean(cid:10)S.E.isshown(n(cid:5)3).Statisticalanalyseswereperformedusingtwo-factorANOVA,withTukey’s
HSDposthoctestsusedforindividualcomparisons.Comparisonsfoundtobestatisticallysignificanttop(cid:9)0.05aremarked(*).hGR-HL,humanGR-L647H/
S651L;hMR-LS,humanMR-H853L/L857S.X,nobindingoractivationobserved.
usingancestralgenereconstructiontoidentifytheobscurecon- as we are left with mesophilic proteins to use for structure-
served structural mechanisms that support activation via functionanalysis.ThisisexemplifiedintheSRfamilyand,in
endogenousversussyntheticligandsthatmaybeexploitedby particular,withmodernGRs,whicharenotoriouslydifficultto
selectivetherapeutics. manipulateunderlaboratoryconditions(23,29).Furthermore,
modernproteinshaveaccumulatedmillionsofyearsofneutral
DISCUSSION
mutationsthatmakeitdifficulttoidentifyfunctionallyimpor-
Wehavesuccessfullyadaptedancestralgenereconstruction
tantaminoacidresidues,aswellasrestrictivemutationsthat
toshedlightonthestructuralmechanismsofdrugselectivity
canfurtherprohibitmutationalanalysis.
for SRs. Our approach combines structural and evolutionary
Workaroundstotheseproblemsarelimitedandfrequently
biology to overcome many of the obstacles that frequently
involve the incorporation of stabilizing mutations. Although
hinderproteinresearchusingmodernproteins.Itiswellknown
this approach does improve the stability of modern proteins,
thatfunction-shiftingaminoacidchangesarenottoleratedwell
including GR (23, 29), mutations such as these may alter the
inmodernproteinsbecausemostproteinsareonlymoderately
wayligandsinteractwiththeirtargetreceptors.Asaresult,the
stable (7, 15, 16). They display a narrow thermal window of
behaviorofthesemutantsmaynotaccuratelymirrorthebehav-
activitydictatedbytheeffectsofnaturalselectiononbothther-
iorofthewild-typeproteins.Incontrast,ancestralproteinsare
malandkineticstability(15)andbytheaccumulationofneutral
subjectedtorigoroustestingduringthereconstructionprocess
mutationsoverevolutionarytime(7).Afinebalanceisneces-
toensuretheirbehaviorisconsistentwiththebehaviorofother
sarytoallowsmallperturbationsorsignals,suchasligandbind-
ing, to functionally alter protein structure: although too little proteinswithintheirphylogeny(e.g.thatthestructuralmech-
stability prevents proper protein folding, too much stability anisms for activation are conserved). Ancestral proteins are
prevents a receptor from adopting an active conformation in inherently more tolerant to mutation and may serve as ideal
responsetostimuliwithinthehostorganism.Wearetherefore models in which to study structure-activity relationships for
limitedbytheeffectsofbothnaturalselectionandneutraldrift, moderatelystableeukaryoticproteins(8,22,23).Evenwhenthe
MAY11,2012•VOLUME287•NUMBER20 JOURNALOFBIOLOGICALCHEMISTRY 16273
DecipheringGlucocorticoidCross-pharmacologyUsingAncSRs
3. Remick,R.A.(1988)Anticholinergicsideeffectsoftricyclicantidepres-
santsandtheirmanagement.Prog.Neuropsychopharmacol.Biol.Psychia-
try12,225–231
4. Decalmer,P.B.,Chatterjee,S.S.,Cruickshank,J.M.,Benson,M.K.,
andSterling,G.M.(1978)Beta-blockersandasthma.Br.HeartJ.40,
184–189
5. Hanania,N.A.,Singh,S.,El-Wali,R.,Flashner,M.,Franklin,A.E.,Garner,
W.J.,Dickey,B.F.,Parra,S.,Ruoss,S.,Shardonofsky,F.,O’Connor,B.J.,
Page,C.,andBond,R.A.(2008)Thesafetyandeffectsofthebeta-blocker,
nadolol,inmildasthma:anopen-labelpilotstudy.Pulm.Pharmacol.Ther.
21,134–141
6. Roy,M.,Dumaine,R.,andBrown,A.M.(1996)HERG,aprimaryhuman
ventriculartargetofthenonsedatingantihistamineterfenadine.Circula-
tion94,817–823
7. Taverna,D.M.,andGoldstein,R.A.(2002)Whyareproteinsmarginally
stable?Proteins46,105–109
8. Bridgham,J.T.,Ortlund,E.A.,andThornton,J.W.(2009)Anepistatic
ratchetconstrainsthedirectionofglucocorticoidreceptorevolution.Na-
ture461,515–519
9. Carson-Jurica,M.A.,Schrader,W.T.,andO’Malley,B.W.(1990)Steroid
receptorfamily:structureandfunctions.Endocr.Rev.11,201–220
10. Stahn,C.,Löwenberg,M.,Hommes,D.W.,andButtgereit,F.(2007)Mo-
lecularmechanismsofglucocorticoidactionandselectiveglucocorticoid
receptoragonists.Mol.Cell.Endocrinol.275,71–78
11. Madauss,K.P.,Stewart,E.L.,andWilliams,S.P.(2007)Theevolutionof
progesteronereceptorligands.Med.Res.Rev.27,374–400
FIGURE6.SchematicsummarizingrelevantfeaturesofSRLBPthatdic- 12. Thornton,J.W.(2001)Evolutionofvertebratesteroidreceptorsfroman
tateMOFactivation.Activationoccurswhenaligand(e.g.MOF(cyan))binds ancestralestrogenreceptorbyligandexploitationandserialgenomeex-
totheLBP,stabilizingtheAF-2helix(darkred)toallowforcoactivatorbinding
pansions.Proc.Natl.Acad.Sci.U.S.A.98,5671–5676
(e.g.TIF2(darkgreen))andsubsequenttranscriptionalcontrol.InGRsandPRs,
13. Bledsoe,R.K.,Montana,V.G.,Stanley,T.B.,Delves,C.J.,Apolito,C.J.,
theLBPcanexpandtoaccommodatesteroidsthataresubstitutedatC3(blue)
orC17(cid:3)(lightgreen).InMR,weidentifiedasinglesiteoutsideoftheLBPthat McKee,D.D.,Consler,T.G.,Parks,D.J.,Stewart,E.L.,Willson,T.M.,
cantoggleMOFagonismversusantagonism,ostensiblybyformingabridge Lambert,M.H.,Moore,J.T.,Pearce,K.H.,andXu,H.E.(2002)Crystal
betweenH7(bluedot)andtheH5-(cid:2)1loop(reddot).Inwild-typeMR,His-853 structureoftheglucocorticoidreceptorligand-bindingdomainrevealsa
makesaweakhydrogenbondthatcannotsupportMOFactivation(redH);the novelmodeofreceptordimerizationandcoactivatorrecognition.Cell
historicalsubstitutiontoapositivelychargedarginine(greenR)strengthens
110,93–105
thisinteraction,restoringactivation.
14. Gee,A.C.,andKatzenellenbogen,J.A.(2001)Probingconformational
changesintheestrogenreceptor:evidenceforapartiallyunfoldedinter-
resurrectionofanentireproteinisnotfeasible,theinsertionof
mediate facilitating ligand binding and release. Mol. Endocrinol. 15,
ancestralresiduesinmodernproteinscanincreasestabilityand
421–428
enhanceadaptabilityandtolerancetomutations(30).Inaddi-
15. Godoy-Ruiz,R.,Ariza,F.,Rodriguez-Larrea,D.,Perez-Jimenez,R.,Ibarra-
tion, we have found that ancestral proteins tend to be more Molero,B.,andSanchez-Ruiz,J.M.(2006)Naturalselectionforkinetic
promiscuoustosyntheticligandsordrugactivation,especially stabilityisalikelyoriginofcorrelationsbetweenmutationaleffectson
incasesinwhichtheancestralproteinsdisplayamorepromis- proteinenergeticsandfrequenciesofaminoacidoccurrencesinsequence
alignments.J.Mol.Biol.362,966–978
cuous phenotype than the extant proteins for endogenous
16. DePristo,M.A.,Weinreich,D.M.,andHartl,D.L.(2005)Missenseme-
ligands.Thus,resurrectedproteinsmaypermitthecrystalliza-
anderingsinsequencespace:abiophysicalviewofproteinevolution.Nat.
tionandfunctionalanalysisofpreviouslyintractablecomplexes Rev.Genet6,678–687
duetotheirenhancedstabilityandpromiscuity. 17. Gaucher,E.A.,Thomson,J.M.,Burgan,M.F.,andBenner,S.A.(2003)
We have shown that, by mirroring what has been done in Inferringthepaleoenvironmentofancientbacteriaonthebasisofresur-
evolutionarystudiesaimedatdiscoveringthestructuralmech- rectedproteins.Nature425,285–288
18. Jermann,T.M.,Opitz,J.G.,Stackhouse,J.,andBenner,S.A.(1995)Re-
anism that conferred hormone selectivity, ancestral proteins
constructingtheevolutionaryhistoryoftheartiodactylribonucleasesu-
maybeusedtoexaminecross-pharmacologyamonghomolo-
perfamily.Nature374,57–59
gous proteins. The advantages of using ancestral proteins to 19. Wouters,M.A.,Liu,K.,Riek,P.,andHusain,A.(2003)Adespecialization
study the structural mechanisms of drug promiscuity lie not stepunderlyingevolutionofafamilyofserineproteases.Mol.Cell12,
onlyintheirenhancedstabilitybutalsoinlocatingthestruc- 343–354
20. Carroll,S.M.,Ortlund,E.A.,andThornton,J.W.(2011)Mechanismsfor
turalfeaturesthatcontributetodifferencesinligandrecogni-
theevolutionofaderivedfunctionintheancestralglucocorticoidrecep-
tion.Ancestralgenereconstructionthereforeprovidesanele-
tor.PLoSGenet.7,e1002117
gantsolutiontosomeofthetroublingproblemsthatcurrently
21. Carroll,S.M.,Bridgham,J.T.,andThornton,J.W.(2008)Evolutionof
interferewiththeprocessofdrugdesign. hormonesignalinginelasmobranchsbyexploitationofpromiscuousre-
ceptors.Mol.Biol.Evol.25,2643–2652
REFERENCES
22. Ortlund,E.A.,Bridgham,J.T.,Redinbo,M.R.,andThornton,J.W.(2007)
1. Nagy,L.,andSchwabe,J.W.(2004)Mechanismofthenuclearreceptor Crystalstructureofanancientprotein:evolutionbyconformationalepis-
molecularswitch.TrendsBiochem.Sci.29,317–324 tasis.Science317,1544–1548
2. Kallenberger,B.C.,Love,J.D.,Chatterjee,V.K.,andSchwabe,J.W.(2003) 23. Bridgham,J.T.,Carroll,S.M.,andThornton,J.W.(2006)Evolutionof
Adynamicmechanismofnuclearreceptoractivationanditsperturbation hormone-receptor complexity by molecular exploitation. Science 312,
inahumandisease.Nat.Struct.Biol.10,136–140 97–101
16274 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER20•MAY11,2012
DecipheringGlucocorticoidCross-pharmacologyUsingAncSRs
24. Tan,R.A.,andCorren,J.(2008)Mometasonefuroateinthemanagement A.,andGreene,G.L.(2007)Structuralplasticityintheestrogenreceptor
ofasthma:areview.Ther.Clin.RiskManag.4,1201–1208 ligand-bindingdomain.EMBORep.8,563–568
25. Bledsoe,R.K.,Madauss,K.P.,Holt,J.A.,Apolito,C.J.,Lambert,M.H., 28. Biggadike, K., Bledsoe, R. K., Hassell, A. M., Kirk, B. E., McLay, I. M.,
Pearce,K.H.,Stanley,T.B.,Stewart,E.L.,Trump,R.P.,Willson,T.M., Shewchuk,L.M.,andStewart,E.L.(2008)X-raycrystalstructureofthe
andWilliams,S.P.(2005)Aligand-mediatedhydrogenbondnetwork novelenhancedaffinityglucocorticoidagonistfluticasonefuroateinthe
required for the activation of the mineralocorticoid receptor. J. Biol. glucocorticoid receptor ligand-binding domain. J. Med. Chem. 51,
Chem.280,31283–31293 3349–3352
26. Madauss, K. P., Deng, S. J., Austin, R. J., Lambert, M. H., McLay, I., 29. Seitz,T.,Thoma,R.,Schoch,G.A.,Stihle,M.,Benz,J.,D’Arcy,B.,Wiget,
Pritchard,J.,Short,S.A.,Stewart,E.L.,Uings,I.J.,andWilliams,S.P. A.,Ruf,A.,Hennig,M.,andSterner,R.(2010)Enhancingthestabilityand
(2004) Progesterone receptor ligand-binding pocket flexibility: crystal solubilityoftheglucocorticoidreceptorligand-bindingdomainbyhigh
structures of the norethindrone and mometasone furoate complexes. throughputlibraryscreening.J.Mol.Biol.403,562–577
J.Med.Chem.47,3381–3387 30. Yamashiro,K.,Yokobori,S.,Koikeda,S.,andYamagishi,A.(2010)Im-
27. Nettles,K.W.,Bruning,J.B.,Gil,G.,O’Neill,E.E.,Nowak,J.,Guo,Y., provementofBacilluscirculans(cid:2)-amylaseactivityattainedusingthean-
Hughs,A.,Kim,Y.,DeSombre,E.R.,Dilis,R.,Hanson,R.N.,Joachimiak, cestralmutationmethod.ProteinEng.Des.Sel.23,519–528
MAY11,2012•VOLUME287•NUMBER20 JOURNALOFBIOLOGICALCHEMISTRY 16275