Epistasis Among Adaptive Mutations in Deer Mouse Hemoglobin

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Epistasis Among Adaptive Mutations in Deer Mouse HemoglobinChandrasekhar Natarajan et al.Science 340, 1324 (2013);

DOI: 10.1126/science.1236862

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MDC12C12011LandCoverTypedatawereobtainedthroughtheonlineDataPoolattheNASALandProcessesDistributedActiveArchiveCenter,U.S.GeologicalSurvey/EarthResourcesObservationandScienceCenter,SiouxFalls,SouthDakota(https://http://wendang.chazidian.comgs.gov/get_data).M.A.Z.acknowledgessupportfromNSFAGS-0840732andAGS-1036275,M.D.acknowledgesaNASAEarthandSpaceScienceGraduateFellowship,andC.H.T.acknowledgessupportfromtheNASARadiationSciencesProgramawardnumbersNNX07AL11GandNNX08AH57G.ThisresearchwassupportedbytheNASAEarthScienceDivisionAtmosphericCompositionprogramawardnumberNNH11AQ58UI.Authorcontributions:single-particleMS,EM,dataanalysis,andpaperwriting(D.J.C.);counterflowvirtualimpactordevelopment,massspectrometerdevelopment,single-particleMS,dataanalysis,andpaperwriting(K.D.F.);compilationofINASdensitiesfromlaboratorydata,derivingthemodel-basedestimatesofuppertroposphericIN,andpaperwriting(C.H.);measurementofwatervapormixingratio,analysisofrelativehumiditydata,andpaperwriting(J.B.S.,M.A.Z,andM.D.);missionplanning,dataanalysis,andpaperwriting(E.J.J);TC4instrumentdesignanddataacquisition,paperwriting

(C.H.T.);andmassspectrometerdevelopment,single-particleMS,dataanalysis,andpaperwriting(D.M.M.).

SupplementaryMaterials

http://wendang.chazidian.com/cgi/content/full/science.1234145/DC1MaterialsandMethodsFigs.S1toS4TablesS1toS4References(33–71)

17December2012;accepted18April2013Publishedonline9May2013;10.1126/science.1234145

EpistasisAmongAdaptiveMutationsinDeerMouseHemoglobin

ChandrasekharNatarajan,1NorikoInoguchi,1RoyE.Weber,2AngelaFago,2HideakiMoriyama,1JayF.Storz1*

Epistaticinteractionsbetweenmutantsitesinthesameprotein

canexertastronginfluenceonpathwaysofmolecularevolution.Weperformedproteinengineeringexperimentsthatrevealed

pervasiveepistasisamongsegregatingaminoacidvariantsthatcontributetoadaptivefunctionalvariationindeermousehemoglobin(Hb).AminoacidmutationsincreasedordecreasedHb-O2affinitydependingontheallelicstateofothersites.StructuralanalysisrevealedthatepistasisforHb-O2affinityandallostericregulatorycontrolisattributabletoindirectinteractionsbetweenstructurallyremotesites.Theprevalenceofsignepistasisforfitness-relatedbiochemical

phenotypeshasimportantimplicationsfortheevolutionarydynamicsofproteinpolymorphisminnaturalpopulations.

onadditiveinteractionsbetweenmuta-tions(epistasis)canexertastrongin-fluenceontherateanddirectionof

evolutionarychange(1,2).Insightsintomecha-nismsofepistasisbetweenbeneficialmutationscanrevealthecausesofconstraintsonadaptiveproteinevolution(3–10).Mechanismsofepis-tasisareoftenbestrevealedthroughdetailedex-aminationsofinteractionsbetweenaminoacidmutationsinthesameproteinthatcontributetovariationinameasurablebiochemicalphenotype

SchoolofBiologicalSciences,UniversityofNebraska,Lincoln,NE68588,USA.2Zoophysiology,DepartmentofBioscience,AarhusUniversity,DK-8000Aarhus,Denmark.*Correspondingauthor.E-mail:jstorz2@unl.edu

1

N

(7,9–15).Suchstudiesareespeciallyrelevanttoourunderstandingofevolutionaryprocesswhengeneticallybasedchangesinthemeasuredphe-notypecontributetovariationinfitnessundernaturalconditions.

Weinvestigatedthenatureofepistaticin-teractionsbetweenadaptivemutationsinthehemoglobin(Hb)ofdeermice(Peromyscusmaniculatus).DeermicethatarenativetohighaltitudehaveevolvedanelevatedHb-O2affinityrelativetolowlandconspecifics(16–18),andthismodificationofproteinfunctioncontributestoanadaptiveenhancementofwhole-animalphys-iologicalperformanceunderhypoxia(19,http://wendang.chazidian.comparisonsbetweenhighlanddeermicefromtheRockyMountainsandlowlanddeermice

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fromtheGreatPlainsrevealedgeneticdiffer-encesinHb-O2affinitythatareattributabletotheindependentorjointeffectsof12aminoacidpolymorphisms:8mutationsinthea-chainsub-unitsofthea2b2Hbtetramer,and4mutationsintheb-chainsubunits.These12aminoacidpoly-morphismsexhibitpronouncedaltitudinalshiftsinallelefrequency,andpopulationgeneticanaly-sesofnucleotidevariationinthea-andb-globingenesrevealedevidencefordivergentselectionbetweendeermousepopulationsthatarenativetodifferentelevations(18,21–23).

StructuralvariationindeermouseHbhasamodularorganizationthatreflectsthelinkagearrangementofthe12aminoacidpolymor-phisms.Withinthea-chainsubunit,fiveaminoacidreplacementsarelocatedinexon2oftheunderlyinggene,andtheremainingthreere-placementsarelocatedinexon3.Polymorphicsiteswithinthesameexonareinnearlycompletelinkagedisequilibrium(LD)withoneanother,butintragenicrecombinationhasproducedapar-tialuncouplingbetweenthetwoexons(21,23).Thetwomostcommona-globinalleleclassesaredistinguishedfromeachotherbyeightaminoacidreplacementsatsites50,57,60,64,71,113,115,and116(fig.S1A).Thefouraminoacidpolymorphismsintheb-globingenearealsoinnearlycompleteLDwithoneanother(18,22).Thetwomostcommonb-globinalleleclassesaredistinguishedfromeachotherbyfouraminoacidreplacementsatsites62,72,128,and135(fig.S1B).Thus,indeermousepopulations,mostofthenaturallyoccurringvariationinHb

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structureiscapturedbycombinatorialpermu-

tationsofallelicvariantsatthreeloci:a-globinexon2,a-globinexon3,andb-globin.

Weusedsite-directedmutagenesistoengi-neeralleightcombinationsofthea-andb-chainvariantsinrecombinantHb(rHb),andwemea-suredO2-bindingpropertiesofthepurifiedpro-teins(24).Inadditiontothechimericmultipointmutants,wealsoengineered10additionalsingle-anddouble-mutantrHbstomeasurethefunction-aleffectsofspecificpointmutationsindividuallyandinpairwisecombination.WesynthesizedrHbsrepresentingthetwomostcommonvar-iantsfromhigh-andlow-altitudepopulations,designated“HH-H”and“LL-L,”respectively(thefirsttwolettersdenotetheseparatea-chainsubdomainsencodedbyexons2and3,andthe

thirdletterdenotestheb-chainsubunit).Totestforepistasis,wealsosynthesizedrHbsrepresent-ingtheremainingsixcombinationsofH-andL-typeallelesateachofthethreeloci(Fig.1A).ToexaminevariationintheallostericregulationofHb-O2affinity,wemeasuredO2-bindingprop-ertiesofeachrHbmutantinthepresenceandabsenceofthetwoprincipalallostericeffectorspresentinmammalianredbloodcells:Cl–ionsand2,3-diphosphoglycerate(DPG).Theseeffec-torsreduceHb-O2affinitybypreferentiallybind-ingandstabilizingdeoxyHb,therebyshiftingtheallostericequilibriuminfavorofthelow-affinityT-statequaternarystructure.ByusingstandardizedconcentrationsofCl–andDPGinthephysiolog-icalrange,weensuredthatinvitromeasurementswererelevanttoinvivoconditions(24).

Ourexperimentsrevealedsubstantialvaria-tioninintrinsicHb-O2affinity,asP50values(theO2tensionat50%hemesaturation)forstripped,cofactor-freerHbsrangedfrom4.55to7.09torr(Table1).Thehigh-altitudeHH-Hvariantex-hibiteda26%lowerstrippedP50(i.e.,higherintrinsicO2affinity)relativetothelow-altitudeLL-Lvariant(Table1).Hb-O2affinitywasre-ducedinthepresenceofCl–ions(addedas0.1MKCl),inthepresenceofDPGatatwofoldmolarexcessovertetramericHb,andinthesimulta-neouspresenceofbotheffectors(Table1).AllrHbsexhibitedcooperativeO2binding,andHillcoefficientsrangedfrom1.36to2.28inthepresenceofCl–andDPG.

Contrarytotheexpectationsofanadditivenullmodel,thephenotypiceffectsofallelicsub-stitutions(L→HandH→L)ata-globinexon2,a-globinexon3,andb-globinwerehighlyde-pendentongeneticbackground,aspairwiseepis-tasisaccountedfor40%ofthevarianceinP50valuesintheabsenceofallostericeffectorsand90%inthesimultaneouspresenceofCl–andDPG(Table1).Inthepresenceofbothallostericeffectors,theHHa-globinalleleconferredanin-creasedaffinityonthebLbackgroundandade-creasedaffinityonthebHbackground.Similarly,theH-typeb-globinalleleconferredanincreasedaffinityontheaLLbackgroundandadecreasedaffinityontheaHHbackground.Theseareex-amplesofsignepistasis(2),wherethesignofthephenotypiceffectofanalleleisconditionalongeneticbackground.

BecausemammalianHbisaheterotetramer(a2b2),epistaticinteractionscouldinvolvecloselylinkedsitesinthesamegeneorsitesinunlinkedgenesthatencodedifferentsubunitsofthepro-tein.Intragenic(within-subunit)epistasiscouldstemfromlocalizedmodificationsofsecondaryortertiarystructure,whereasintergenic(between-subunit)epistasiscouldstemfromallosterictran-sitionsinquaternarystructurebetweendifferentoxygenationstatesoftheHbtetramer.EpistasisforHb-O2affinityismainlyattributabletothesuppressedDPGsensitivityofchimericrHbvar-iantsthatincorporatetheproductsofa-andb-globinallelesofunliketype(aHHcombinedwithbL,andviceversa;Table1andFig.1B).AlthoughDPGsensitivitywassuppressedin

Fig.1.StructuralandfunctionalvariationamongrecombinantdeermouseHbs(rHbs).(A)rHbsrepresentingallcombinatorialpermutationsofallelicvariantsata-globinexon2,a-globinexon3,andb-globin.Shadedregionsrepresenttheproductsoflow-altitudeL-typealleles(red),andunshadedregionsrepresentproductsofhigh-altitudeH-typealleles(blue).(B)Variationintheallostericregula-tionofHb-O2affinitybyDPG.SensitivitytoDPGisindexedbythedifferenceinlog-transformedP50valuesbetweenstrippedHbinthepresenceandabsenceofDPG.

Table1.O2affinities(P50,torr;meanTSEM)andallostericpropertiesofpurifiedrHbs.Sensitivitiestoallostericeffectorsaremeasuredasthedifferenceinlog-transformedP50valuesinthepresenceandabsenceofeacheffector,individuallyandincombination.VAandVEareestimatedcomponentsofadditiveandepistaticvariance,respectively(24).

LL-L

P50(torr)stripped+KCl+DPG

+KCl+DPG logP50

KCl strippedDPG stripped

(KCl+DPG) stripped

5.7210.209.7210.46

TTTT0.230.030.170.30

HL-L6.3712.559.6411.21

TTTT

LH-L

HH-L6.5912.887.548.63

TTTT0.110.270.250.21

LL-H5.2512.156.937.65

TTTT0.090.310.190.22

HL-H5.4911.188.8411.36

TTTT

LH-H

HH-H4.559.709.5310.13

TTTT0.080.470.270.35

VA0.5970.0510.1890.096

VE0.4030.9490.8110.904

0.057.09T0.550.4110.83T0.780.1210.33T0.200.3312.51T0.59

0.1840.1630.247

0.046.32T0.150.3012.01T0.200.2611.44T0.300.3412.83T0.34

0.2790.2580.308

0.2510.2300.2620.2950.1800.2460.2910.0580.1170.3640.1210.1640.3090.2060.3160.3290.3210.3480.6710.3290.7260.2740.2140.786

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HH-LandLL-H,allostericregulatorycapacitiesandfig.S2).Additionalhydrogenbondsbe-increaseinHb-O2affinityindividuallyorincom-ofthechimericrHbswerepartiallyrestoredbytweensubunitsofthesameabdimerareformedbination;however,ontheHH-Hbackground,reciprocallyconvertingeitherofthetwoa-chaininthepresenceofb128Ser(anL-typeresidue;single-stepreversionstoL-typeresiduesatbothsubdomainstothetypethatmatchedtheasso-fig.S2),whichcontributestotheobservedepis-sitesproducedsignificantreductionsinHb-O2ciatedb-chainsubunit:DPGsensitivityofthetasisbetweenallelica-andb-chainvariants.affinityinthepresenceofallostericeffectorschimericLL-HwaspartiallyrestoredbyL→HStructuralanalysisalsorevealedthatinHbswith(fig.S3).Wealsomeasuredtheindividualeffectssubstitutionsata-globinexon2orexon3,andL-typea-globin,theimidazoleringofa50Hisofallfouraminoacidmutationsintheb-chainreciprocally,DPGsensitivityofthechimericformsahydrogenbondwitha30Gluinthesamesubunit.OntheLL-Lbackground,thesubsti-HH-LwaspartiallyrestoredbyH→Lsubstitu-subunit.Thereplacementofa50HiswithPro(thetutionb128Ser→Ala,whichremovesana1b1tionsatthesesameloci(Fig.1B).Inprinciple,aH-typeresidue)eliminatesthishydrogenbondhydrogenbond(fig.S2),producedanincreasedsuppressedDPGsensitivity(andhence,increasedandcausesasubtlereorientationoftheEhelixanionsensitivity(andhence,adecreasedHb-O2Hb-O2affinity)couldbeproducedbycharge-andCDloop(Fig.2),aneffectthatpropagatesaffinityinthepresenceofCl–andDPG;fig.S4).changingaminoacidreplacementsthateliminatetothea1b2intersubunitcontactandshiftstheHowever,onthissamebackground,introducingphosphate-bindingsitesintheb-chainsubunits.allostericequilibriuminfavorofthehigh-affinityallfourH-typeb-chainmutationsincombina-Becausethepositivelychargedphosphate-bindingoxyHb(R-state)quaternarystructure.

tionproducedahighlysignificantincreaseinsitesareinvariantindeermousebchains(17,18),Totesttheeffectsofcharge-changinga-chainHb-O2affinityinthepresenceofallostericef-allelicvariationinDPGsensitivitymuststemfrommutationsintheCDloop(a50His/Pro)andthefectors(fig.S4).

indirect,second-orderperturbations.

adjacentEhelix(a64Asp/Gly),wesynthesizedInsummary,resultsofourmutagenesisex-AnalysisofthecrystalstructureofdeereachofthealternativesingleanddoublemutantsperimentsrevealedpervasiveepistasisamongmouseHbat1.8Åresolution(24,25)revealedonbothHH-HandLL-Lbackgrounds.Theex-segregatingaminoacidvariantsindeermousethateachoftheeightrHbmutantsischarac-perimentsrevealedthat,ontheLL-Lbackground,Hb(Table1).TheindividualandjointeffectsterizedbyauniqueconstellationofhydrogensubstitutionsofH-typeresidues(a50His→Proofa-andb-chainpointmutationscontributetobondswithinandbetweensubunits(Table2

anda64Asp→Gly)didnotproduceasignificant

theelevatedHb-O2affinityofhighlanddeermice,buttheeffectsofthesemutationsarehighlyTable2.Allelicvariationinthenetworkofatomiccontactswithinandbetweensubunitsofdependentontheallelicstateofotherresiduedeerpositions.

a113His-mousea24Tyr)Hb.Plusorbetweensignsdenotesubunitstheofpresenceunliketypeofhydrogen(a34Cys-bbonds128Ser).withinPolymorphicsubunitssites(a50His-areshowna30Gluinbold.

andDirectedmutagenesisstudieshaveunveiled“LL-L

HL-L

LH-LHH-L

LL-HHL-H

LH-HHH-H

tionscrypticthat”distinguishepistasisbetweendeeplydivergedaminoacidorthologoussubstitu-H-bonds

proteins(7,11,13,14).Similarly,experimentalastudiesofmicrobialsystemshaverevealedin-

a50His113His-a-30Glua++tragenicepistasisbetweensitesthatunderwenta34Cys-b128Ser

24Tyr+

++++

++

+

+

+

successiveallelicsubstitutionsbutthatwereneversimultaneouslypolymorphic(6,10).Bycontrast,theinteractingmutationsindeermouseHbaresegregatinginnaturalpopulationsand,giventheextensiveintragenicandintergenicLD,theepistasiscontributestoadditivegeneticvarianceinHbfunction,providinganexplanationforthepreviouslydocumentedvariationinanionsensitivityofdeermouseHbs(17,18).GiventheevidenceforspatiallyvaryingselectiononHbpolymorphisminrelationtoaltitude,theperva-sivenessofsignepistasisforHb-O2affinitysug-geststhattheselectioncoefficientforagivenallelewilloftenbehighlydependentonthealleliccompositionofthelocalpopulation.Thus,signepistasisamongsegregatingaminoacidvariantsmayexertastronginfluenceonallelefrequencydynamicsandmutationalpathwaysofproteinevolution.

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http://wendang.chazidian.commun.69,393(2013).Acknowledgments:FundedbygrantsfromtheNIH–NationalHeart,Lung,andBloodInstitute(R01HL087216and

HL087216-S1),theNSF(DEB-0614342andIOS-0949931),andtheFacultyofScienceandTechnology,AarhusUniversity.WethankS.Kachmanforstatisticaladvice,A.Bangforassistanceinthelab,andM.Harms,S.Smith,andtworeviewersforhelpfulcomments.Allexperimentaldataaretabulatedinthemaintextandinthesupplementarymaterials.

SupplementaryMaterials

http://wendang.chazidian.com/cgi/content/full/340/6138/1324/DC1MaterialsandMethodsFigs.S1toS5TableS1

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21February2013;accepted8April201310.1126/science.1236862

JodieL.Rummer,1,2*DavidJ.McKenzie,3AlessioInnocenti,4ClaudiuT.Supuran,4ColinJ.Brauner1TheRooteffectisapH-dependentreductioninhemoglobin-O2carryingcapacity.Specifictoray-finnedfishes,theRooteffecthasbeenascribedspecializedrolesinretinaloxygenationandswimbladderinflation.Wereportthatwhenrainbowtroutareexposedtoelevatedwatercarbondioxide(CO2),redmusclepartialpressureofoxygen(PO2)increasesby65%—evidencethatRoothemoglobinsenhancegeneraltissueO2deliveryduringacidoticstress.Inhibiting

carbonicanhydrase(CA)intheplasmaabolishedthiseffect.WearguethatCAactivityinmusclecapillariesshort-circuitsredbloodcell(RBC)pHregulation.ThisacidifiesRBCs,unloadsO2fromhemoglobin,andelevatestissuePO2,whichcoulddoubleO2deliverywithnochangeinperfusion.ThispreviouslyundescribedmechanismtoenhanceO2deliveryduringstressmayrepresenttheincipientfunctionofRoothemoglobinsinfishes.nvertebrates,hemoglobin(Hb)playsacru-cialroleinoptimizingtissueoxygen(O2)deliverybyincreasingbloodO2-carryingca-pacityandregulatingthepartialpressure(PO2)atwhichO2isdelivered.Withintissues(suchasmuscle),metabolicallyproducedcarbondioxide(CO2)reducesbloodpHandthusHb-O2affin-

I

ity,elevatingbloodPO2andenhancingO2deliv-ery,whichiscollectivelytermedtheBohreffect(1).Inmammals,thismayelicitanincreaseinbloodPO2ofupto2mmHg(2,3)invivo,pro-vidingsome5%increaseinO2delivery.TeleostfishesoftenhavemuchmorepH-sensitiveHbs,andarecentinvitrostudyindicatesthatthisef-

DepartmentofZoology,UniversityofBritishColumbia,6270UniversityBoulevard,Vancouver,BritishColumbiaV6T1Z4,Canada.2AustralianResearchCouncilCentreofExcellenceforCoralReefStudies,JamesCookUniversity,Townsville,Queensland4811,Australia.3EquipeDiversitéetEcologiedesPoissons,UMR5119EcologiedesSystèmesMarinsCôtiers,UniversitéMontpellierII,PlaceEugèneBataillon,Case093,F-34095Montpelliercedex5,France.4UniversitàdegliStudidiFirenze,NeurofarbaDepartment,SezionediScienzeFarmaceutiche,ViaUgoSchiff6,50019SestoFiorentino(Florence),Italy.*Correspondingauthor.E-mail:

jodie.rummer@jcu.edu.au

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Fig.1.(AandB)Schematicrepresentationofacatecholamine-activatedRBCpHdisequilibrium(A)short-circuitedbyplasma-accessibleCAtoelevatetissuePO2(B).AE,anionexchange;cAMP,adenylatecyclaseand3′,5′-cyclicmonophosphate.

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RootEffectHemoglobinMay

HaveEvolvedtoEnhanceGeneralTissueOxygenDelivery

fectmaybeanorderofmagnitudegreater(4).Thecurrentstudyconfirmsthisinvivo.EnhancedO2deliverymayrepresentanimportantstepintheextraordinaryadaptiveradiationoftheteleostfishes(5),whichmakeupalmosthalfofallver-tebratespecies.

Inteleosts,areductioninbloodpHreducesbothHb-O2affinityandO2carryingcapacity,whichisknownastheRooteffect(6)andhasawell-studiedroleinsecuringO2deliverytotheretinaandswimbladder(7,8).Thesetissuespos-sessspecializedacid-producingcellsinconjunc-tionwithadensecounter-currentcapillarynetwork(rete)thatlocalizesandmagnifiesalargeacidosis,thuspromotingO2-offloadingviatheRooteffect(7,8).ThissystemiseffectiveenoughtogenerateO2tensionsexceeding50atm(~38,000mmHg)withinthegas-filledswimbladder(7).