1

StructuralStudiesofaProtocadherin-15FragmentEssentialforHearing

AthesispresentedBy

ConghuiChenTo

TheCommitteeonDegreesinChemistryadBiochemistry

InpartialfulfillmentoftherequirementsForadegreeof

BachelorofSciencewithResearchDistinctionInthefieldofBiochemistry

ResearchAdvisor:Dr.MarcosSotomayor,AssistantProfessorofDepartmentofChemistryandBiochemistryDefenseCommittee:Dr.JohnShimko,ChemistryLecturerofDepartmentofChemistryandBiochemistry

TheOhioStateUniversity

Columbus,Ohio

April13th,2016

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StatementofResearch

Iconductedtheresearchpresentedinthisthesisundertheprofessionalguidanceof

Dr.MarcosSotomayorofTheOhioStateUniversityMainCampusChemistryand

BiochemistryDepartment.IjoinedtheSotomayorlabinAugustof2013,duringmy

secondyearattheuniversity.Iwastrainedintheprocessofproteinpurificationand

cellculturebyvisitinggraduatestudentDeryanurKilicinconjunctionwithDr.

Sotomayor.Dr.Sotomayorofferedknowledgeabletutelageindesigningand

performingtheexperiments,analyzingdata,andthewritingofthisthesis.All

molecularmodelingandanalysiswasperformedwiththeguidanceandassistance

fromDr.MarcosSotomayorandDr.RaulAraya-Secchi.Myresearchwasgenerously

fundedbyTheOhioStateUniversityChemistryandBiochemistryUndergraduate

ResearchScholarshipfromautumn2013untilspring2015.Iperformedresearchas

partoftheBiochemistry4998and4999coursesasarequirementforthe

completionofthethesis.

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Abstract

Soundtravelsthroughtheexternalandmiddleeartothefluid-filledcochleawhere

mechanosensitivehaircellstransformitintoelectrochemicalsignals.Ontheapical

sideofeachhaircell,asetofhair-likeprotrusions,calledstereociliaformabundle

withfilamentousconnections(tiplinks)thatareessentialforhearing.Inresponse

tothemechanicalforcegeneratedbysoundswaves,stereociliamovebackandforth,

therebystretchingtiplinksandopeningnearbytransductionchannels.Thetiplink

isformedbytwonon-classicalcadherins,Cadherin-23(CDH23)andprotocadherin-

15(PCDH15),whicharemembersofthecadherinsuperfamilyofcalcium-

dependentadhesionproteins.CDH23has27extracellularcadherin(EC)repeatsand

PCDH15contains11ECrepeats.SinglemissensemutationsinPCDH15areknownto

causedeafness,andabsenceofthisproteinleadstobothdeafnessandblindness.A

recentstudyshowedthatapointmutation(V767-)locatedinthePCDH15EC7repeat

ispathogenic.MyprojectfocusesondeterminingthestructureofthePCDH15EC7to

EC8fragmentviaX-raycrystallography,bothtounderstanditsfunctioninhearing

andtodiscoverthestructuraleffectsofpathogenicmutations.

InafirststeptoachievethesegoalsweusedaDNAconstructthatencodesforthe

mousePCDH15EC7-8fragmenttoexpressitinabacterialsystem.After

transformationandculture,wesuccessfullyobservedproteinexpression.However,

theproteinfragmentwasnotsolubleandaggregatedininclusionbodies.Therefore,

Ididproteinpurificationunderdenaturingconditions,followedbyrefoldingand

size-exclusionchromatography,andobtainedpurefoldedproteinamenablefor

crystallization.IobtainedproteincrystalsthatdiffractedwhenexposedtoX-rays

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andthenrefinedthecrystallizationconditionstoobtainafulldatasetwitha

resolutionof2.0Å.UsingthisdatasetIfoundamolecularreplacementsolutionfor

thefirstcrystalstructureofPCDH15EC7-8,whichIamcurrentlyrefining.Inthis

thesisIwilldescribeeachofthestepsinvolvedinobtainingthestructureofPCDH15

EC7-8andwilldiscusshowthestudyofthisstructurewillhelpusunderstandthe

mechanismsofmechanotransductioninnormalandimpairedhearing.

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AcknowledgmentsFirstandforemost,Iwouldliketoextendthesincerestofthankstomythesis

advisorDr.MarcosSotomayorforalloftheeffortsthathehasputintomakingmy

timeinhislaboneofthemostrewardingandexcitingexperiencesofmylife.Dr.

Sotomayor’sdedicationandcommitmenttoeducationandscienceinfluenceme

everytimeweinteract.Nomatterhowbusyorcrazyhisscheduleis,hewillalways

answerallofmyquestionsandoffersteadilyhelpwithallsupport.More

importantly,healwaysencouragesmetotakechallengesandraisequestionsto

perfectmetoabetterperson.Itistrulymypleasureandprivilegetobeoneofhis

students,andIhopethatonedayIwillbeabletoliveuptotheexamplethathehas

setformeoverthepastthreeyears.

IwouldalsoliketoextendthankstoCarissaKlanseckforcollaboratingwithmeon

thisproject.Sheisathoughtfulandgenerousformerundergraduatestudentand

gavemesupportthatallowedmetobeabletoachievetheresultspresentedinthis

thesis.

IwouldalsoliketogivehugethankstoDr.RaulAraya-Secchi,thepost-doctoral

fellowthatIworkedwithalongsidefor2years.Iwouldliketothankhimforhis

generoussupportofsharinghisstructurewithmesothatIcanachievemyfinal

structure.Heisapersonfullofknowledgeandhelpfulforassistingmewithany

technicalissueswheneverIconfrontany.

Iwouldliketothankallofmygenerousandkindresearchco-workersinthe

Sotomayorlab.Itisyourendlesssupportandhelpthatmakesmetobemetoday.

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Besidesworkinginthelab,wealsoarebecominglifelongfriendswitheachother,

whichisaverypreciousexperienceandtimetome.

Lastbutnotatleast,IwouldliketogivethankstoTheOhioStateUniversityfor

offeringmescholarshipsforthepasttwoyearthatgavemeinspirationandsupport

duringschoolyears.

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TableofContentsStatementofResearch...........................................................................................................2Abstract......................................................................................................................................4Acknowledgements................................................................................................................6FiguresIndex............................................................................................................................8TablesIndex..............................................................................................................................8Chapter1:Introduction........................................................................................................91.1 Hearing,mechanotransduction,andthetiplink…..............................................121.2 Thecadherinsuperfamilyofproteins...................................................................141.3 TiplinkinteractionsandstructureofCDH23andPCDH15…….......................141.4 PCDH15EC7-8……......................................................................................................15

Chapter2:Producingtheprotein....................................................................................162.1Cloning,propagation,andsequencingofmmpcdh15EC7-8..............................182.2ProteinexpressionofPCDH15EC7-8......................................................................192.3Purificationandone-steprefoldingofPCDH15EC7-8........................................202.4PurificationofPCDH15EC7-8...................................................................................21Chapter3:CrystallizationandDataCollection...........................................................223.1CrystallizationofPCDH15EC7-8..............................................................................243.2RefineofPCDH15EC7-8crystallizationconditions..............................................253.3DiffractiondatacollectionofPCDH15EC7-8..........................................................283.4DataprocessingandstructuredeterminationoftheEC7-8crystals.................30Chapter4:StructureandInterfaceAnalysis...............................................................314.1Calciumbindingsiteincadherins..............................................................................334.2AnalysisofPCDH15EC7-8structure........................................................................334.3Locationofdeafnesssite..............................................................................................354.4ApotentialPCDH15parallelinterface......................................................................384.5Theimportanceofstudyingnon-classicalcadherins.............................................39References...............................................................................................................................43

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FiguresIndexChapter11.Schematicofthehumanear.............................................................................................102.Anatomyoforganofcorti.................................................................................................113.Mechanotransductioninvertebratehaircells.............................................................124.CalciumionsimpactC-cadherinequilibrium...............................................................135.RibbondiagramofPCDH15EC1-2interactwithCDH23EC1-2………………...........146.PCDH15EC7-8.....................................................................................................................15Chapter27.ProteinexpressionresultofPCDH15EC7-8.................................................................188.ProteinpurificationanalyzedbySDS-PAGE.................................................................199.Processofsize-exclusionchromatography...................................................................2010.Size-exclusionchromatographyoflarge-scaleproteinpreparations...................21Chapter311.Protein-crystallizationviasittingdropvapordiffusionmethod.............................2312.Proteincrystalsinpre-screenPEGsuit.......................................................................2413.RefinementscreenforPCDH15EC7-8crystallization..............................................2514.ProteincrystalforX-raycrystallographyanddiffractionpattern.........................2515.DiffractionofPCDH15EC7-8.........................................................................................2616.StructureandtopologydiagramofPCDH15EC7-8..................................................29Chapter417.SequencealignmentofPCDH15extracellularrepeats.............................................3218.MutatedsequenceinPCDH15EC7...............................................................................3419.PointmutationwithinPCDH15EC7calciumbindingmotifs..................................3420.PointmutationshowninCPDH15EC7........................................................................3521.PotentialinterfacesofPCDH15EC7-8.........................................................................37TablesIndexChapter31.Diffractiondataindex........................................................................................................28Chapter42.InterfaceofPCDH15EC7-8predictedbyPISA.............................................................36

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Chapter1:Introduction

Hearing,astheabilitytoperceivesoundbydetectingvibrations,isconsideredtobe

oneofthemostimportantsensationsinall-livingorganismsandinhumans.

However,deafnessisfoundtobethemostcommonlossofperceptionasitaffects

morethan40millionpeopleintheUnitedStates1.Deafness,anaetiological

heterogeneoustrait,iscausedbyvariousgeneticandenvironmentalfactors2.Itdoes

notonlyhaveagreatinfluenceinone’slife,butalsoimpactsthewholefamily3.

Hearinglosshasbeenobservedinupto20%of10year-oldchildren4.Unfortunately,

itisoftenhardtodiagnoseandtreatauditorydiseasebecausethesensoryhaircells

donothavetheabilitytoregenerate3,5.

Potentialhearinglosstreatmentshavebeendiscovered,suchaspartialdeafness

treatment(PDT),whichutilizesbothelectricalandacousticstimulationtoenable

hearing6.Nonetheless,ourknowledgeofhearingimpairmentisverylimitedand

genetictreatmentsforhearinglosshavenotbeensuccessfullydevelopedyet.To

elucidatethemechanismofsomeformsofhereditarydeafness,wewanttotakean

in-depthlookatthestructureofanon-classicalcadherin,protocadherin-15

(PCDH15),whichiscrucialforauditorymechanotransduction7,8.Therefore,Iwill

firstintroducetheroleofPCDH15innormalhearingandthebasicsofthecadherin

familyinChapter1.Next,IwilldiscusshowIproducedandpurifiedafragmentof

PCDH15(EC7-8)inChapter2.InChapter3,Iwillillustratetheprocessofprotein

crystallizationanddatacollection.Finally,Iwillanalyzethepreliminarystructureof

protocadherin-15EC7-8,itscalcium-bindingmotifs,itsmutationsitesrelatedto

deafness,anditspossibleinterfaces.

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1.1Hearing,mechanotransduction,andthetiplink

Soundtravelsfromtheouterearthroughtheexternalauditorycanaltothe

tympanicmembranecausingittovibrate.Theeardrumthenpropagatesthe

vibrationtotheossicles,wherethesoundenergyistransmittedovertotheoval

windowattheentranceofthecochlea(Fig.1).Thecochlea,aspiral-shaped,fluid-

filledorganembeddedinthetemporalbone,housesthebasilarandtectorial

membranesandtheorganofcortiwheremechanotransductiontakesplace.The

soundenergycausesoscillationofspecificregionsofthebasilarandtectorial

membranes(Fig.2).

Figure1:Theexternalearcontainstheauricle,earcanal,andtympanicmembrane.ThemiddleearcontainstheossiclesandisconnectedtothepharynxbytheEustachiantube.Theinnerearcontainsthecochleaandvestibule,whichareresponsibleforauditionandequilibrium,respectively.From32.

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Ontopofthebasilarmembrane

istheorganofcorti,which

containssensitivereceptorcells

calledinnerandouterhaircells.

Ontheapicalsideoftheinner

haircellsitsthestereocilia

bundle(Fig.3a),whichis

responsiblefordetectingfluid

vibrationinthecochlearduct9.

Thestereociliatipsofouterhaircellsareembeddedinthetectorialmembraneand

detectmovementofthebasilarmembranerelativetothetectorialmembrane.The

stereociliarowsarrangeinorderofincreasingheightandareconnectedtoeach

otherbyafilamentousstructurecalledthetiplink10(Fig.3b-d).Mechanicalforce

fromsoundcausesthestereociliatodeflect,therebystretchingthetiplinksand

openingnearbytransductionchannels11.Whenstereociliabendsinthedirectionof

thetalleststereocilium,tiplinksstretch;mechanotransductionionchannelsthen

openandallowpotassiumionstoenterthecell.Theinfluxofpotassiumionscauses

thecelltodepolarizeandgenerateactionpotentialsinafferentneurons.When

stereociliabundlebendsinthedirectionoftheshorteststereocilium,tiplinksslack;

mechanotransductionionchannelsthencloseandstopthepotassiumcurrent.The

cellthenhyperpolarizesandneurotransmitterreleasedecreasesorstops.Thetip

linkconsistsoftwoproteins:Protocadherin-15(PCDH15)andCadherin-23

Figure2:Anatomyoforganofcorti.From33.

BasilarmembraneSpiralganglion

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(CDH23),whichareessentialforhearingandbelongtothecadherinsuperfamilyof

proteins.

Figure3:Mechanotransductioninvertebratehaircells(a)Haircellstererociliaaligninincreasingheighttowardthetalleststereocilia,whichdefinestheexcitatoryaxis.(b)TiplinkstructureconsistsofCDH23andPCDH15andconnectsthetopofonestereociliatothesideofitstallestneighbor.(c)Stereociliadeflectduringmechanotransduction.(d)Acloseuplookofthetip-linkregionwithionchannelslocatedatthelowerendofeachtiplink.Adaptedfrom30.

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1.2Thecadherinsuperfamilyofproteinsandtheroleofcalcium

Thecadherinsuperfamilyincludesclassicalcadherins,aswellasclusteredandnon-

clusteredprotocadherins,allwithextracellularcadherin(EC)repeats12,13.Cadherins

canbeseenasasignificantcomponentofvariouslivingmulticellularorganisms,

whichplaysanimportantroleincellsignalingandmechanicalprocesses13.The

structureofcadherinhasbeenrecognizedasEC“repeats”thatpositioninseries

withcalciumionsinbetweentheECrepeatsthatmodulatetheirelasticityand

mediateitscell-celladhesionability14.Thefunctionofcalciumions,whichsupport

thestructuralintegrityofcadherin’slinkerregions,hasbeenstudiedusing

biochemicalandcomputationaltools15–17.

Theresultshowsthattheextracellulardomainiscapableofmaintainingitscrystal

conformationwithcalciumionsbyforminganelongatedandcurvedstructure,

Figure4:CalciumionsimpactC-cadherinconformation.SimulationsofthecompleteC-cadherinextracellulardomainwithandwithoutCa2+ions,respectively(AandB).Theproteinisshownincartoonrepresentationanditssurfaceisdrawnintransparentorange.GreenandredspheresarecrystallographicCa2+andterminalCαatoms.ScreenshotsofthecompleteextracellulardomainofC-cadherinafter10nsofequilibrationinthepresenceofCa2+ions(C),intheabsenceofCa2+ions(D),respectively.WhileC-cadherinremainscurvedwhensimulatedinthepresenceofCa2+,itsshapeislostafter10nsofdynamicsintheabsenceofCa2+.Adaptedfrom16.

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whileitsstructureisflexiblewithoutthepresenceofcalciumions16.Thus,calcium

ionsareessentialforcadherintoestablishandmaintainitsproperconformation.

1.3TiplinkinteractionsandstructureofCDH23andPCDH15tipsHaircellsarethemechanosensorsresponsiblefortransducingmechanicalforces

originatedfromsoundwavesandheadmovements1.Haircellsthenconvertthe

mechanicalforcesintoelectrochemicalsignals,whichcanbesenttothebrain

throughtheauditorynerve1.Themechanoelectricaltransductionchannellocates

nearthetipsofstereocilia,wheretheextracellulartip-linkfilamentsarelocated18,19.

Thisfinefilamentstructureiscomposedoftwoproteins,CDH23andPCDH15,

whosemutationsareknowntocausedeafness20,21,7.

Crystallographyandmoleculardynamicssimulations

studieshavebeenconductedtodeterminehowCDH23

bindstoPCDH15.Aunique“handshake”modelhasbeen

foundtobethecadherininteractionmechanism,in

whichanantiparallelheterodimerbetweenthetwo

mostamino-terminalcadherinrepeats(ECrepeats1

and2)ofeachproteinisformed22.

Thecomplexiscalcium-dependent,astheseions

providerigidityandfacilitatethehandshakeinteraction.

Multiplemissensemutationsatcalciumbindingsites

andatthehandshakeinterfaceareknowntocause

deafness7,22.

Figure5:RibbondiagramofPCDH15EC1+2(purple)interactingwithCDH23EC1+2(blue)withCa2+ionsasgreenspheres.Adaptedfrom22.

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1.4PCDH15EC7-8

PCDH15isoneofthenon-classicalmembersofthecadherinsuperfamilythathave

multipleECrepeats.Therehavebeenvaststudiesfocusingonclassicalcadherins,

butweonlyhavelimitedknowledgeaboutthenon-classicalcadherins,suchas

PCDH15.Toourknowledge,thestructureofPCDH15EC4-11hasneverbeen

discovered.Thus,bystudyingtheEC7-8,itcouldallowustohaveafurtherinsightof

thiscadherincomplex.

Figure6:CartoondisplayofPCDH15ECrepeatswithEC7-8markedinred.

BasedonthestructureofPCDH15EC1-2,wepredictthatPCDH15EC7-8isabout

200aminoacidsinlength,whichweighsabout24kDa.ApointmutationinEC7is

knowntocausenon-syndromicdeafness23.Inaddition,wealsofindaninteresting

variationinEC7atoneofitscalcium-bindingmotifs(DYE).Resolvingthestructure

ofPCDH15EC7-8willallowustounderstandtheroleofthedeafnessmutationin

EC7,thestructuralimplicationsofthesequencevariationinEC7’scalcium-binding

motifs,andalsopossibleparalleldimersofPCDH15.

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Chapter2:Producingtheprotein

Hearingisextremelyimportantforvertebratelife,andherewefocusonthe

structuresoftiplinkproteinsthatareessentialforinner-earmechanotransduction.

Studyingthestructureofprotocadherin-15(PCDH15)fragmentsthatformpartof

thetiplinkcouldallowustohaveabetterunderstandingofitsroleinhearing

mechanotransduction.Ourgrouphasstudiedthetip-linkinteractioninvolvingsmall

PCDH15andCDH23tips,butthewholestructureofPCDH15hasnotbeensolved

yet.Thus,ourgrouphasdesignedvariousconstructstoexpressandpurify

fragmentsofPCDH15.IworkedwithoneofthemencodingforMusmusculus

PCDH15EC7-8,anduseditforexpression,purificationandcrystallizationas

describedinthischapter.

2.1Cloning,propagation,andsequencingofmmPCDH15EC7-8

TheMusmusculusPCDH15cDNAwasusedasthesourceforcloningPCDH15EC7-8

intothepET21avector(cloningperformedbyDr.Sotomayorandco-workers).The

fragmentconstructwasdesignedbyusingthepublishedstructureofPCDH15EC1-2

asareference(ProteinDataBankcode4APX).Dr.Sotomayordesignedprimersthat

allowedamplificationofthedesiredfragmentandincludedthesequenceofNdeI

andXhoIrestrictionenzymesites.AfteramplificationusingPCR,therestriction

enzymesNdeIandXhoIwereusedtodigesttheamplifiedsequence(PCRproduct),

whichwasthenligatedtoadigestedplasmidvectorpET21a(+).ThepET21vector

carriesanampicillinresistancegene,anisopropylβ-Dthiogalactopyranoside(IPTG)

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inducibleT7promoter,andaC-terminalsix-histidinetagtoallowforNi-affinity

purificationoftheexpressedgenefragment.IusedDH5αE.colicellsandstandard

“miniprep”protocolstopropagateandobtainmoremmPCDH15EC7-8DNAplasmid.

ThelengthoftheinsertwasverifiedbyNdeIandXhoIenzymedigestionsand

agarosegelanalysis.Inaddition,theplasmidwassequenceverifiedwiththeT7

promoterandT7-terminatorprimers.

2.2ProteinexpressionofPCDH15EC7-8

BL21-Gold(DE3)E.colicellswereusedtoexpressthePCDH15EC7-8construct.

ThesecellscontaintheT7polymerasegeneandhavebeenmodifiedforhigh

efficiencyproteinproduction.AllBL21cellsweregrowninLysogenyBroth(LB)

medium.

Totestforproteinexpression,singlecoloniesoftransformedBL21cellswere

inoculatedandgrownovernight.Then,asmallamountof25mLexpressionculture

with100µg/mLofampicillinwasinoculatedwith200µloftheovernightculture

andincubatedat37°CuntiltheculturereachedanOD600of0.6,atwhichpointIPTG

wasaddedforafinalconcentrationof200µM.Thecellsweregrownat37°Cwith

shakingovernight,andwerethenpelletedbycentrifugation.Asmallsampleof

pelletswasresuspendedinSDSloadingbufferandboiledfordenaturation.The

samplewasthenloadedontoaSDS-PAGEgelandstainedwithCoomassieto

determinetheexpressionoftheproteinfragmentofinterest.Proteinexpressionfor

mmPCDH15EC7-8wassuccessful(Fig.7).ThePCDh15EC7-8proteinwasthen

expressedatalargerscaleforfurtherbiochemicalassessmentsandcrystallization

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assays.A2Llargeculturewasinoculated

with25mLofovernightBL21cellculture.

Theliquidmediumwaspreheatedto37°C

foroptimalgrowthpriortotheadditionof

thecellculture.Thelargeculturewasthen

grownuntiltheOD600reached0.6,induced

withIPTG(200µMfinalconcentration),and

incubatedovernightat37°C.Onthe

followingday,thecellswerecollected,

pelleted,andstoredat-20°C.

2.3Purificationandone-steprefoldingofPCDH15EC7-8

Protocadherinshaveatendencytoforminclusionbodies.Thus,PCDH15fragments

werepurifiedunderdenaturingconditions.Sonicationwasfirstusedtolysethe

pelletedcellswithadenaturingbindingbuffer(20mMTris-HClpH7.5,10mM

CaCl2,6Mguanidiniumhydrochloride,and20mMimidazolepH7.5).Roughly35

mLofbindingbufferwasaddedto6gofcellpellet.Thelysatewasthencentrifuged

at20,000rpmfor30minutesat4°Ctowashoffanycellulardebris.After

centrifugation,theprotein-richsupernatantwasincubatedwithNi-sepharosehigh

performancebeadsforonehourat4°C.Afterincubation,themixturewas

centrifugedagainat3,000rpmfor5minutesat4°Candthesupernatantwas

decanted.TheNibeadswerewashedagainandthemixturewasthenplacedinthe

Figure7:ProteinexpressionresultofPCDH15EC7-8at37oCat0hrvs.overnight.MolecularweightofPCDH15EC7-8is~24kDa,markedinred.

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denaturingelutionbufferwithhighimidazole

concentration(20mMTris-HClpH7.5,10mM

CaCl2,6Mguanidiniumhydrochloride,and500

mMimidazolepH7.0)toelutetheprotein(Fig.

8).

TorefoldPCDH15EC7-8,theproteinelution

wasdilutedwithdenaturingelutionbuffertoa

concentrationof<0.5mg/mL.Thediluted

proteinwasdialyzedat4°Covernightusing

bufferD(20mMTris-HClpH8.0,5mMCaCl2,

150mMKCl,5mMCaCl2,10%Glycerol),which

dilutesguanidiniumhydrochloridefromthe

proteinsolutiontopromoterefoldingoftheproteinfragment.Wefoundthat

glycerolwasabletoassistPCDH15EC7-8’srefoldingbypreventingprotein

precipitation.Afterovernightdialysis,theproteinsolutionwascollectedand

centrifugedat20,000rpmfor30minutesat4°Ctoremoveprecipitations.After

centrifugation,theproteinsolutionwasconcentratedwitha10,000MWCOfilter

(polyethersulfonemembrane)toconcentrationsrangingbetween3to9mg/mLfor

futuresize-exclusionchromatography(SEC)andsubsequentproteincrystallization.

Figure8:ProteinpurificationresultanalyzedbySDS-PAGE.Firstlane:Proteinladder.Secondlane:flowthrough.Thirdlane:firstwashwithbindingbuffer.Fourthlane:secondwashwithbindingbuffer.Fifthlane:Proteinelution.MolecularweightofPCDH15EC7-8:24kDa.

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Figure9:Processofsize-exclusionchromatography(SEC).(A)Schematicpictureofabeadwithanelectronmicroscopicenlargement.(B)Schematicdrawingofsamplemoleculesdiffusingintobeadpores.(C)Graphicaldescriptionofseparation:(i)sampleisappliedonthecolumn;(ii)thesmallestmolecule(yellow)ismoredelayedthanthelargestmolecule(red);(iii)thelargestmoleculeiselutedfirstfromthecolumn.(D)Schematicchromatogram.Adaptedfrom34.

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2.4PurificationofPCDH15EC7-8

TheproteinwasfurtherpurifiedbySEC(Figs.9&10)withaS200Superdex16/60

GL(GEHealthcare)columnandbuffercontaining20mMTrisHClpH8.0,150mM

KCland5mMCaCl2.Thismethodseparatesproteinbasedonsize,astheporous

resinofthecolumntrapsanddelaysproteinswithlowhydrofluidicvolumewhile

largerproteinsbypasstheporesandareelutedoutfaster.Theconcentratedprotein

solutionwasfilteredbeforeloadingontothecolumnbyusinga0.45µmPES

membrane.AfterSEC,IusedCoomassie-stainedSDS-PAGEtodeterminethepurity

andintegrityoftheelutedfractions(Fig.10B).

Figure10:Size-exclusionchromatographyresultofrefoldedPCDH15EC7-8(A)andSDS-PAGEgelanalysisofpeaks(B).

A B

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Chapter3:CrystallizationandDataCollectionProteincrystallizationcanbeachievedundersuitableconditionsinwhichprotein

moleculesformrepeatingpatternsbynon-covalentinteractionswithintheprotein

crystal24.Therearenumerousvariablesthatcanaffectthesensitivityofprotein

crystallizationability,suchastemperature,ionicstrength,proteinconcentration

andpH25.Additionally,thegrowthofproteincrystalsalsorequiresdifferenttime

periodsanditisnearlyimpossibleforustopredictexactcrystallizationconditionsa

priori.

Inthischapter,IwillexplainhowIcrystallizedPCDH15EC7-8andhowIdesigneda

refinementscreentosearchforgooddiffractingcrystals.Aftersuccessful

crystallization,weshotthecrystalswithX-rays,obtainedacompletedataset,and

usedmolecularreplacementtodeterminethePCDH15EC7-8structure.Ourfinal

modelat2.0ÅresolutionshowsforthefirsttimethearchitectureoftheseEC

repeats,includingcanonicalcalcium-bindingsitesatthelinkerbetweenEC7and

EC8andthelocationofsitesthataremutatedinhereditarydeafness.

3.1CrystallizationofPCDH15EC7-8

TocarryoutX-raycrystallographyonPCDH15EC7-8,Iexpressedandpurifiedthis

proteinfragmentasdescribedinchapter2.Theproteinfragmentswerethen

concentratedto4mg/mLand7mg/mL,anddispensedona96-wellsittingdrop

tray,whichcontainedalargerwellforreservoirsolutionandasmallerwellfor

purifiedproteinmixedwithreservoirsolution(Fig.11).TheproteinfragmentEC7-8

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wascrystallizedbysittingdropvapordiffusionmethod,whichrequiredboththe

reservoirbufferandpurifiedproteintobewithinaclosedsystem.

Thesittingdroputilizedtheprincipleofvapordiffusionbetweenthereservoir

bufferandpurifiedproteinsolution,wherewaterinthepurifiedproteinsolution

vaporsintothereservoirbufferinordertobalanceconcentrationandreachan

optimalconditionforproteincrystallization26,27.Atfirst,Iusedthreedifferentbuffer

suitestodeterminethebestconditionsformyproteinconstruct’scrystallization:

QiAGENclassics,PEG(polyethyleneglycol),andcationsuites.Iadded75μLof

reservoirbufferintothebiggerwell,0.6μLofpurifiedproteinsolutionintothe

smallerwell,andtransferred0.6μLofreservoirbufferintothesmallerwell.The

trayswerethensealedwithplastictapeandstoredat4oC.Theproteincrystallized

approximatelyafteronemonthofsettingupthetrays.Icloselymonitoredprotein

growthundertheopticalmicroscopeduringthismonth.Theproteincrystallizedin

severalofthePEGsuiteconditionsattheconcentrationof4mg/mL(Fig.12).

Figure11:Proteincrystallizationbysittingdropvapordiffusionmethod.Adaptedfrom35.

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Dr.MarcosSotomayor“fished”theproteincrystalwitha0.05mmloopandshotthe

proteincrystalwithX-raysusingthehomesourceatthefirstflooroftheRiffe

building(RigakUMicroMax003).Thediffractionpatternindicatedthatwehada

proteincrystal,sotheproteincrystalwassavedinliquidnitrogenforfutureanalysis

atAPS(AdvancedPhotonSource)intheArgonneNationalLaboratory.

3.2RefinementofPCDH15EC7-8crystallizationconditions

Basedontheproteincrystallizationconditionmentionedabove,Idesignedmy

refinementscreentoprovideanoptimalsetofconditionsforcrystalgrowth(Fig.

13).ThecrystalsIobservedthatwerebestforX-raydiffractionweregrownin

ammoniumchloridewithPEG3350,ammoniumsulfatewithPEG3350,MESwith

PEG20000,andammoniumsulfatewithglycerolandPEG3350.Iuseddifferent

concentrationsofPEG3350,PEG20000,ammoniumchlorideandammonium

sulfate,aswellasMESatvariouspH.

Figure12:Proteincrystalsinpre-screenPEGsuiteunderdifferentconditions.

25

Aftermonitoringthegrowthofproteincrystalsinthisrefinement,Isuggestedthat

theproteingrewbestinammoniumchlorideandPEG3350,wherethebest

conditionsareshowninredinFigure13.

ThesecrystalswerefishedbyDr.MarcosSotomayorandsenttoAPS(Advanced

PhotonSource)atArgonneNationalLaboratory.

Figure13:RefinementscreenforPCDH15EC7-8crystallization.

Figure14:ProteincrystalsofPCDH15underrefinementconditions.

26

3.3DiffractiondatacollectionofPCDH15EC7-8 ThediffractiondataforEC7-8crystalswerecollectedonthebeamlineofthe

AdvancedPhotonSource(APS)atArgonneNationalLaboratory.Dr.Marcos

Sotomayorassistedmetoindex,integrateandscalethediffractiondatawithHKL

2000.

Figure15:DiffractionofPCDH15EC7-8.PCDH15EC7-8crystals(Figure13-B2)grewinarefinementconditioncontaining10%ammoniumchlorideand0.2MPEG3350anddiffractedtohighresolutionandexhibitednosignsoftwinning,makingstructuredeterminationfeasible.ThediffractionimageisthedatasetthatwasusedtodeterminethestructureoftheEC7-8fragment.

27

Thebestdiffractiondatawegotwasundertheconditionof0.2Mammonium

chlorideand10%PEG3500withfinalresolutionof2.0Å.Thespacegroupwe

observedwasC121.Preliminarystatisticsfordatacollectionandrefinementare

showninTable1below.

28

Datacollection MmPCDH15EC7-8Spacegroup C121Unitcellparameters a,b,c(Å) 135.796,22.99,70.741α,β,γ(°) 90,97.268,90Moleculesperasymmetricunit 1Beamsource APS-24-ID-CWavelength(Å) 0.9792Resolutionlimit(Å) 2.000Uniquereflections 14026Completeness(%) 96.5(82.1)Redundancy 3.1(2.4)I/σ(I) 5.4Rmerge 0.045Rmeas 0.054Rpim 0.030CC1/2 (0.974)CC* (0.993)Refinement Resolutionrange(Å) 50.00-2.00(2.03–2.00)Rwork(%) 0.1796Rfree(%) 0.2414Residues(atoms) 212(1575)Watermolecules 110Rmsdeviations Bondlengths(Å) 0.0173Bondangles(°) 1.8342B-factoraverage 41.78Protein 41.80Ligand/ion 34.94Water 41.52RamachandranPlotRegion(PROCHECK) Mostfavored(%) 87.9Additionallyallowed(%) 10.3Generouslyallowed(%) 0.0Disallowed(%) 0.0Table1:PreliminarystatisticsfordatacollectionofPCDH15EC7-8.Thenumberspresenttheinformationofcompletedataset.Thenumbersinparenthesesindicatesthehigh-resolutionshell.

29

3.4DataprocessingandstructuredeterminationforPCDH15EC7-8

ThemacromolecularmodelofPCDH15EC7-8wasbuiltusingCoot(Crystallographic

Object-OrientedToolkit)andaninitialmodelofPCDH15EC8-10byDr.

RaulAraya-Secchi.WemadeuseofthePCDH15EC8modeltodomolecular

replacement,obtaininitialphases,andbuildthewholeEC7-8structure.

Figure16:(A)StructureofPCDH15EC7-8visualizedbyVMD.(B)TopologydiagramofPCDH15EC7-8.Themissingresiduesareindicatedinred.

EC7

EC8

A B

30

Aswebuiltthemodel,weusedthesoftwareRefmactodoreciprocalspace

refinement.ThecurrentR-factoris0.18andtheRfreefactoris0.24,whichindicate

theagreementbetweenourcrystallographicmodelandtheexperimentaldata. The

relativelysmallRfactorof0.18impliesaminordissimilaritybetweenexperimental

observationsandtheoreticalcalculatedvalues.

31

Chapter4:StructureAnalysisThestudiesinthisthesisrepresenttheinitialstagesofthestructural

characterizationofPCDH15EC7-8withitscalcium-bindinglinkerregion.After

optimizingexpression,refolding,andpurificationprotocolforPCDH15EC7-8,I

successfullycrystalizedanddetermineditsstructure.Thestructurerevealsthatthe

linkerregionbetweenEC7andEC8inPCDH15bindsthreeCa2+ions,whichisthe

canonicalbindingmodepreviouslyobservedinthemajorityofcadherinstructures.

Inthischapter,Iwillanalyzethiscanonicalcalciumdependentstructureandits

calciumbindingsites.Next,IwilltalkaboutthesitewithinPCDH15thatisknownto

causedeafnesswhenmutated.ThenIwilldiscussinterestinginterfacesthatare

formedbycrystalcontactsofPCDH15EC7-8.Finally,Iwilldiscusstheimplications

oftheresultsobservedinthisstudy,andproposefurtherexperimentsthatare

aimednotonlyatreproducingtheseresults,butalsoatgainingabetter

understandingofnon-classicalcadherins.

4.1Calciumbindingsitesincadherins

Therearefiveconservedcalcium-bindingmotifsbetweentheECrepeatsin

cadherinsingeneral.Thecompletesequencealignmentofthe11ECrepeatsof

PCDH15isshownbelow(Fig.17)andshowsthepresenceorabsenceofthesemotifs

inthisprotein28.

32

Figure17:AlignmentofPCDH

15ECrepeatsofinterestforthisthesis.Mutationslinkedto

deafnessarecircledinred.Aminoacidm

otifsformingcalcium

-bindingsitesareshownabove

alignment(XEX

BASE,DXD,DRE,XDX

TOP,DXNDN).Adaptedfrom

28.

33

Therearefivecalcium-bindingmotifswithinPCDH15extracellularrepeats.Thefirst

bindingmotifistheXEXBASE,thesecondoneistheDXD,thethirdoneistheDRE,the

fourthoneistheXDXTOPandthelastoneisDXNDN.Theletter“X”indicatesanynon-

conservedaminoacids,whilethepreservedaminoacid,suchastheEinthefirst

calcium-bindingmotif,indicatesitsinteractionwiththecalciumionswithinthe

repeats.Thosefivecalcium-bindingmotifsworktogethertomaintainthestructure

ofPCDH15andtodetermineitselasticity.

4.2AnalysisofPCDH15EC7-8structure

Basedonourmolecularreplacementsolutionandrefinement,weobtainedthe

molecularstructureofPCDH15EC7-8(Fig.16),whichissimilartothestructureof

othercadherinmolecules.TheEC7-8fragmentcontains209aminoacidsintotal,

whichcorrespondtoresidues692to897.Wewereabletoplacethemajorityofthe

aminoacids,watermoleculesandthreecalciumionsintheelectrondensity.ChainA

containsmajoraminoacidsofourproteinfragment,chainBcontainscalciumions

andchainCcontainswatermolecules.Therewerestillafewmissingresidues

startingfromresidue863to867thatwewereunabletoplacebecauseofthelackof

electrondensity.Accordingtothemolecularstructure,therewerethreecalcium

ionsbetweenEC7andEC8.ThefirstcalciumiscoordinatedbyGlu708,Asn757and

Glu759,thesecondcalciumiscoordinatedbyGlu759,Asp790,Asn794andGlu826

andthethirdoneiscoordinatedbyAsp792,Asp824andAsp826.Thestructureof

EC7-8isconservedandsimilartothatofPCDH15EC1-222.

34

4.3Locationofdeafnesssite

PCDH15,essentialfortheauditorysystem,isknownasanon-classicalcadherin

withalargeextracellulardomainthatisinvolvedinhereditarydeafness.Themost

frequentcauseofblind-deafnessinhumans,Ushersyndrome,isrelatedtogenetic

mutationsthatmodifyPCDH15amongotherproteins.Anunusualpointmutation

locatesinPCDH15EC7at

V767-,wherethein-frame

deletionofthisvalineis

knowntocauseinherited

non-syndromicdeafness

DFNB2323.

ThepointmutationlocatesbetweenthethirdCa2+bindingmotif(DYE)andthe

fourthCa2+bindingmotif(XDXTOP)asshownbelow(Fig.19),anddoesnotdirectly

affectcalciumbinding(thepointmutationiscircledinred).

Figure18:Mutatedsequence(V767-)inPCDH15,whichishighlyconservedinmammalianspecies.Adaptedfrom23.

Figure19:PartialsequencealignmentofPCDH15ECrepeatswithpointmutationV767-circledinred.Adaptedfrom31.

35

Figure20:PointmutationinPCDH15EC7visualizedinVMD.

AswehavesolvedthestructureofPCDH15EC7-8,weareabletolocatethepoint

mutationwithinoursequencetoknowhowwouldthemutationaffectthe

crystallizedstructure(Fig.20).Wearealsoabletoknowhowthepointmutation

couldinterferewithinthecrystalcontactsthatmayshowphysiologicallyrelevant

interfaces.

EC1IVVQEC2IIQAEC7VVVAEC8SITF

36

4.4ApotentialPCDH15parallelinterface

AfterfinalizingapreliminarymodelforPCDH15EC7-8,weanalyzeditwith

PDBePISA(ProteinInterfaces,SurfacesandAssemblies)todeterminepossible

intermolecularinterfaces29.PISAsuggeststhreefeasibleinterfacesbetweenEC7-8

units(Table2andFig.21).

Table2:PISAinterfacelistofPCDH15EC7-8.

37

Figure21:PISAanalysis.(A)AnantiparallelinterfaceofPCDH

15EC7-8indicatedbyPISA.(B)AparallelinterfaceofPCDH

15EC7-8indicatedbyPISA.(C)AnantiparallelinterfaceofPCDH

15EC7-8indicatedbyPISA.Thepositionofvalineresidueiscircledinred.

38

Thefirstresultindicatesanantiparallelinterfaceareaof750.6Å2withaΔGP-value

of0.955.ThislargeP-valueindicatesthattheinterfaceislikelytobea

crystallographycontact.Thesecondresultindicatesaparallelinterfacewithan

interfaceareaof741.9Å2andaΔGP-valueof0.148.ThesesmallP-valuesindicate

thattheinterfaceismorehydrophobicthanusualcrystallographiccontactsandmay

existinsolution.Thethirdresultindicatesanantiparallelinterfacewithaninterface

areaof290A2andaΔGP-valueof0.509.ThisP-valueindicatesthattheinterfaceis

norsurprisingintermsofhydrophobicity.Becauseofitsrelativelysmallsurface

areacomparedtotheprevioustworesults,wediscardit.Basedonourpreliminary

result,wearestilldebatingaboutthepotentialparallelandantiparallelinterfacesof

PCDH15EC7-8.Webelievethatdeterminingwhetheranyoftheseinteractionsis

validwouldhelpustohaveabetterunderstandingofPCDH15functionandthe

structureofitsentireextracellulardomain.

4.5Theimportanceofstudyingnon-classicalcadherinsMystructuralanalysisofthePCDH15EC7-8fragmentrevealsthecanonicalcalcium

ionstoichiometryandcoordinationmechanismoftheEC7-8linkerregion.Thepoint

mutationV767-providesusanopportunitytovisualizetheinteractionofthis

deafness-relatedmutationandspeculateaboutitsmechanism23.Interestingly,by

analyzingthecrystalcontacts,wemaybeabletodetectpossibleinterfaces.By

finalizingtheinterfaceanalysis,wecoulddeterminewhetherPCDH15formsa

parallelhomodimerinwhichEC7andEC8formkeycontacts.Tovalidatethis

39

interface,crystalstructureswithmoreECrepeatsareneeded.Ananalysisof

glycosylationsitesisalsoneeded.

40

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