Structure Article The NMDA Receptor NR1 C1 Region Bound to Calmodulin: Structural Insights into Functional Differences between Homologous Domains ZeynepAkyolAtaman,1LokeshGakhar,1BrendaR.Sorensen,1JohannesW.Hell,2andMadelineA.Shea1,* 1DepartmentofBiochemistry 2DepartmentofPharmacology RoyJ.andLucilleA.CarverCollegeofMedicine,UniversityofIowa,IowaCity,IA52242-1109,USA *Correspondence:[email protected] DOI10.1016/j.str.2007.10.012 SUMMARY includingcalmodulin(CaM).CaMhastwohighlyhomolo- gousdomains(NandC);eachdomainhastwoEF-hand Calmodulin (CaM) regulates tetrameric N- sites that bind calcium cooperatively. CaM regulates methyl-D-aspartate receptors (NMDARs) by a widearray of target proteins, including kinases, phos- binding tightly to the C0 and C1 regions of its phatases, and ion channels (cf. Bhattacharya et al., NR1subunit.Acrystalstructure(2HQW;1.96A˚) 2004;ChinandMeans,2000;SaimiandKung,2002;Vet- of calcium-saturated CaM bound to NR1C1 terandLeclerc,2003). Upon calcium influx, CaM induces inactivation of (peptide spanning 875–898) showed that NR1 NMDAR(reducingitsopenrateandmeanopentime)by S890, whose phosphorylation regulates mem- bindingtoNR1C0andC1(Ehlersetal.,1996b).Inactiva- brane localization, was solvent protected, tionreleasesNMDARfromtheneuronalcytoskeletonby whereas the endoplasmic reticulum retention disrupting interactions between NR1 and a-actinin2 motifwassolventexposed.NR1F880filledthe (Krupp et al., 1999; Zhang et al., 1998). CaM binding to CaMC-domainpocket,whereasT886wasclos- NR1C1significantlyenhancesNMDARinactivation(Eh- est to the N-domain pocket. This 1-7 pattern lers et al., 1996b), which absolutely requires C0 (Zhang was most similar to that in the CaM-MARCKS et al., 1998). Dissociation constants for (Ca2+) -CaM 4 complex. Comparison of CaM-ligand wrap- binding to peptides representing the CaM-binding do- around conformations identified a core tetrad mains (CaMBDs) of NR1 showed that its affinity for C1 of CaM C-domain residues (FLMM ) that con- (NR1C1p; aa 875–898) was 20-fold more favorable than C its affinity for C0 (NR1C0p; aa 838–863) (Ehlers et al., tacted all ligands consistently. An identical 1996b).TheK values(4and87nM,respectively)suggest tetradofN-domainresidues(FLMM )madevar- D N that (Ca2+) -CaM binds both C0 and C1 intracellularly, iable sets of contacts with ligands. This CaM- 4 where [CaM] is 50–75 nM (Wu and Bers, 2007). C1 free NR1C1 structure provides a foundation for hasanendoplasmicreticulum(ER)retentionmotif(R893– designing mutants to test the role of CaM in R895;Figure1A)andaProteinKinaseC(PKC)phosphor- NR1traffickingaswellasinsightsintohowthe ylationsite(S896)thatareneededforpropermaturation homologousCaMdomainshavedifferentroles andERrelease(Scottetal.,2001;Standleyetal.,2000). inmolecularrecognition. NR1 S890 phosphorylation by PKC disrupts surface- associatedclustersofNR1,causinganevendistribution throughout fibroblasts (Tingley et al., 1997), and affects INTRODUCTION receptorpotentiation(Zhengetal.,1999).Thepositionof CaM relative to these regulatory motifs in NR1 is not The ionotropic N-methyl-D-aspartate receptor (NMDAR) known. isamajorsourceofcalciumfluxintoneuronsinthebrain ToexploretheroleofCaMinNR1trafficking,itisnot andhasacriticalroleinlearning,memory,neuraldevelop- possible to make a viable CaM knockout organism be- ment, and synaptic plasticity (Mori and Mishina, 1995). causeCaMisessentialandhasmultipletargets.Ourstrat- MammalianNMDARshavetwofamiliesofsubunitsdesig- egy was to determine the binding interface to provide natedNMDAR1(NR1)andNMDAR2(NR2).Understand- aplatformfortargetedmutagenesisofNR1.Here,were- ing the roles of NR1 and NR2 subunits in brain function portacrystalstructureof(Ca2+) -CaMboundtoNR1C1p 4 is complicated by variable developmental and spatial (1.96 A˚ resolution) in which the CaM domains wrapped expressionof theirmRNA,andbythepresence ofeight aroundhelicalNR1C1p(Figures2Aand2B).TheERreten- variants of NR1 arising from N-terminal sequence varia- tionmotif(R893–R895)andS896weresolventexposed, tionsandalternativesplicingoffourexonsencodingthe whereas S890 was buried in the N domain of CaM. C0 (membrane-proximal), C1, C2, and C20 regions (Fig- NR1C1pwaspredictedtobea1-12CaMBD(Yapetal., ure1A).TheC-terminaltailofNR1bindsseveralproteins, 2000), but it was found to be a 1-7 CaMBD. NR1 F880 Structure15,1603–1617,December2007ª2007ElsevierLtdAllrightsreserved 1603 Structure CalmodulinBoundtoNMDARNR1C1Region Figure2. CrystalStructureoftheCaM-NR1C1pComplex (A) NR1C1p sequence and structure superimposed on its electron densitymapcontouredat1.0s. (BandC)AlternateviewsofCaM-NR1C1p(2HQW)showingtheCaM N-domainbackbone(blue),theCdomain(red),Ca2+ionsandbinding sites(yellow),andNR1C1p(gray).ThefigurewasmadewithMacPymol. (DandE)Alignmentof17canonicalCaM-targetcomplexesbytheirCa atomsofthe(D)N-domain(residues5–72;68atoms)and(E)C-domain (residues84–146;63atoms)FLMMresiduesasdescribedinExperi- mentalProcedures. Figure1. CaMBindingtoNMDARNR1 (A)SchematicdiagramofNR1indicatingrelativepositionsofintracel- regulatingMARCKSattachmenttothecytoskeletonand lularregionsC0,C1,andC2/C20,andsequencesofC0andC1.The proteinsbindingC1suggestasimilarmechanismofregu- CaMBDsequenceofC0(residues838–865)showsthesingletrypto- lationofC1intheformationofNR1-richclusters(Ehlers phan(presumedanchor)residueboxed.ThesequenceofC1(residues 875–898)isshownwiththeERretentionsignal(RRR)underlined,the etal.,1996b;Tingleyetal.,1997). PKCsitesboxedandshaded,andresiduesF880andT886boxed. The1-7motiffoundhereisunusualamongCaM-target (B)BindingofCaMtoNR1C1pmonitoredbyfluorescenceanisotropy interfacesin17complexesinwhichboththeNandCdo- ofFl-NR1C1p(intrinsicvalueof0.04)toafinalconcentrationof51.5mM mainsofCaMcontactsthetarget.Theonlyotherknown apoCaM(open;K =158mM)or0.76mM(Ca2+)-CaM(filled;K = D 4 D case is CaM bound to MARCKS. To determine whether 1.99 nM). The asterisk indicates that the anisotropy of Fl-NR1C1p thenatureaswellasthespacingofCaMresiduescritical titratedwithapoCaMwasnormalizedtothevalue(0.13)observed aftersaturationwithcalcium. to molecular recognition were different among these (C) Simulation of apo CaM (dashed black) and (Ca2+)-CaM (solid structures, we analyzed the CaM-target contacts in all 4 black) binding to NR1C1p with equilibrium constants from (B). For 17complexesandfoundthat4residuesintheCdomain comparison,bindingofCaMtoNR1C0p(gray)simulatedwithaKD (F92,L105,M124,andM144:FLMMC)wereusedconsis- of87nM(Ehlersetal.,1996b)for(Ca2+)4-CaM(solid)andaKDof tentlybyCaMtocontacttargets.Althoughastructurally 2.25mM(Akyoletal.,2004)forapoCaM(dashed). equivalenttetrad(F19,L32,M51,andM71:FLMM )was N was anchored in the C domain of CaM, whereas T886 observed in the CaM N domain, these CaM residues (ratherthanF891)contactedthehighestnumberofresi- werenotusedidenticallybyalltargets. duesintheCaMNdomain.Thesame1-7motifandnearly identicalprimarycontactresidueswereidentifiedforCaM RESULTS bound to a MARCKS (myristoylated, alanine-rich, PKC substrate)peptide(1IWQ).Inthatcase,CaMbindinginter- BindingofCaMtoNR1C1pandNR1C0p ruptsattachmentoftheactincytoskeletontotheplasma TitrationsofNR1C1pwith(Ca2+) -CaMandapo(Ca2+-de- 4 membrane (Aderem, 1992). Parallels between proteins pleted)CaM(Figure1B)yieldedaK of2.0±0.1nMfor D 1604 Structure15,1603–1617,December2007ª2007ElsevierLtdAllrightsreserved Structure CalmodulinBoundtoNMDARNR1C1Region (Ca2+) -CaM, which agreed well with the value of 4 nM 4 Table1. CrystallographicDataCollectionand determinedpreviously(Ehlersetal.,1996b).AKDof158± RefinementStatistics 3mM(K of6.33E3M(cid:2)1)wasresolvedforapoCaMbind- A (Ca2+) -CaM-NRC1pCrystal ingtoNR1C1p.ComparedtoNR1C0,(Ca2+) -CaMbinds 4 4 DataCollectionStatistics C0withaK of87nM(Ehlersetal.,1996b)andapoCaM D bindsC0withaKDof2.25mM(Akyoletal.,2004).Compar- Spacegroup P3221 isonofsimulatedequilibriumtitrationsofCaMbindingto Celldimensions a=40.361A˚;b=40.361A˚; NR1C1p andNR1C0p(Figure1C)showedthatthemid- c=175.765A˚;a=90(cid:3); pointsforapoand(Ca2+) -CaMbindingtoNR1Cpdiffered b=90(cid:3);g=120(cid:3) 4 byfourordersofmagnitude. Resolution(A˚) 19.69(cid:2)1.90(2.00(cid:2)1.90) Structureofthe(Ca2+) -CaM-NR1C1pComplex RsymorRmerge 0.034(0.255) 4 The crystal structure of (Ca2+) -CaM bound to NR1C1p I/sI 17.36(3.92) 4 was determined to 1.96 A˚ resolution (Figures 2A–2C). It Completeness(%) 98.4(92.6) adoptedthecanonicalCaM-targetconformationinwhich Redundancy 3.01(2.56) boththeNandCdomainsofCaMcontactedpeptideto formacompact,ellipsoidalcomplex.CaMresidues1,2, RefinementStatistics 75–80, and 148 were disordered and were not included Resolution(A˚) 8.57(cid:2)1.90 inthemodel.Figure2Ashowstheelectrondensitymap Numberofreflections 13,379 for NR1C1p (residues S897 and K898 were disordered); thepeptidewasbuiltintothefinalmodelmanually.Refine- Rwork/Rfree(%) 20.6/24.8 mentstatisticsaregiveninTable1. Bfactorforprotein(A˚) 33.8 This structure was compared to 16 other similar and Bfactorforligand 43.5 nonredundant(Ca2+) -CaM-targetstructures(listedinEx- 4 perimentalProcedures).Toevaluatetheoverallstructural Bfactorforions 31.4 variabilityinthese17compactCaM-targetcomplexes,we Bfactorforwater 41.8 comparedeachonetoanaveragestructureasdescribed Rmsdbondlengths(A˚) 0.019 inExperimentalProcedures.Among17structures,theav- eragermsdoftheCaMNdomainwas0.75A˚,whereasthat Rmsdbondangles((cid:3)) 1.585 oftheCdomainwas0.59A˚ (Figures2Dand2E).Thecom- Numberofproteinatoms 1,089 plexesthatshowedthehighestdeviationfromtheaverage Numberofligandatoms 178 backboneconformationintheCdomainwerethosede- terminedbyNMR(CaMwithCNGchannel[1SY9],CaMKK Numberofions 4 [1CKK],andskMLCK[2BBM]);skMLCKalsohadthehigh- Numberofwateratoms 66 estN-domaindeviation. Twostructures ofthedrugTFP Ramachandranplot(%residues) bound to CaM (1A29 for the N domain; 1LIN for the C domain)hadthesmallestrmsdvalues. Mostfavored 94.2 Additionallyallowed 5.8 AccessibilityofNR1C1pMotifs Disallowed 0.0 Processing and localization of C1-containing NR1 sub- units is regulated by an ER retention motif (R893–R895) Valuesinparenthesesrefertothehighest-resolutionshell. and by phosphorylation of S896. These residues had ahighfractionalsolvent-accessiblesurfacearea(SASA): R893,89.4%;R894,71.6%;R895,92.4%;S896,87.1%; (Figure 4). Among the ordered side chains, this analysis averageSASA,85%(Figure3A).Incontrast,S890(impli- identified 36 residues in the N domain (residues 3–74) cated in subunit clustering and receptor potentiation by and34intheCdomain(residues81–147)thatmetthiscri- PKC) was protected by the N domain of CaM, having terion.AsshowninFigures4Aand4B,contactswiththe only 41% SASA. Two CaM N-domain residues (M36, CaMNdomainwerewelldistributedacrossthelengthof M51)werewithin4.5A˚ ofNR1S890asdeterminedbyus- NR1C1p: 17 with the N-terminal half (residues 875–885; ing Contacts of Structural Units (CSU) (Sobolev et al., gray)and19withtheC-terminalhalf(residues886–896; 1999). The hydroxyl of S890 was 3.22 A˚ from the sulfur black). In contrast, contacts with the CaM C domain ofM36and4.01A˚ fromthatofM51,suggestingthatthese were skewed: 27 with the N-terminal half of NR1C1p residuesinteractinthecomplex(Figure3B).Nocontacts andonly7withtheC-terminalhalf(Figures4Band4C). were observed between NR1 S890 and any residues in While CSU analysis showed that most NR1C1p resi- theCaMCdomain. dues contacted a single CaM domain, side chains of NR1C1p K875, K876, T879, and L887 contacted 2 or InterfaceContacts more residues in each domain. The 7 contacts of K875 ToexploretheCaM-NR1C1pinterface,CSUwasusedto are shown in purple and are underlined in Figure 5A. determineCaMresidueswithin4.5A˚ ofNR1C1presidues NR1C1pF880contactedthehighestnumberofresidues Structure15,1603–1617,December2007ª2007ElsevierLtdAllrightsreserved 1605 Structure CalmodulinBoundtoNMDARNR1C1Region main(i.e.,FLMM andFLMM )appearedtoadoptnearly N C identical spatial conformations (Figure 5C). The electron density overlap of side chains of FLMM residues and C NR1C1pF880isshowninFigure5D.Theperpendicular orientation of NR1C1p F880 relative to CaM F92 allows forafavorablep-pinteractionbetweenthetwoaromatic rings(SinghandThornton,1992). IdentifyingCaMResiduesCommonlyUsed forTargetInteractions ThereisonlyoneotherstructureofCaMboundtoa1-7 CaMBDmotif,buttherearenumerouscompact,ellipsoi- dal CaM-target structures. To explore how the CaM- NR1C1p interface related to those complexes, we used CSU to conduct a statistical analysis of CaM residues contactingtargetsin16othercompact(Ca2+) -CaM-tar- 4 getstructures(12CaM-peptide,4CaM-drugcomplexes; listedinExperimentalProcedures).Inthesetof17struc- tures analyzed, 3 CaM residues (F92, M124, and M144) contacted every target; in all but one structure, L105 alsocontactedthetarget(Figure6A).Thus,FLMM con- C sistentlyservesastheverticesoftheC-domainhydropho- bic pocket in these structures. An overlay of domains alignedaccordingtotheCaatomsoftheFLMM tetrad C shows that the positions of these FLMM residues in all structuresisinFigure6B. AcorrespondinganalysisoftheNdomainshowedthat Figure3. SolventAccessibilityofS890 althoughresiduesintheFLMM tetradwerecontactedin N (A)Surface(CaM)andstick(NR1C1p)diagramof2HQWcoloredas atleast12oftheanalyzedstructures,theseresidueswere agradientfromblue(buried)tored(exposed)accordingto%SASA notthe4residuescontactedmostfrequently(Figure6C). values:S890,41%;R893,89%;R894,72%;R895,92%;andS896, 87%. Instead,E11wastheonlyresiduefoundtobewithin4.5A˚ (B)Ball-and-stickdiagramofS890andCaMN-domainresidues(M36, of the target peptide or drug in all 17 of the structures M51,andQ41). that were examined. However, 2 of those 17 structures ThefigurewasmadewithMacPymol. (1CTR.pdband1A29.pdb)wereCaM-drugcomplexesin which the pocket of the N domain was vacant. Thus, (7)withinasingledomainofCaM(Figure4B).F880was E11 interacted with target molecules bound exclusively within 4.5 A˚ of F92, I100, L105, M124, A128, F141, and in the hydrophobic cleft of the C domain. The second M144 (red letters in Figure 5A). All have hydrophobic mostcommonlyusedN-domainresidue,A15,alsocon- sidechainslocatedintheCdomainofCaM.Thepeptide tactsthetargetassociatedwiththeCdomainofCaM.In residue making the second highest number of contacts the15structuresthathadthehydrophobiccleftoftheN withinasingledomainofCaMwasT886.Itspartnersin domain occupied, F19 contacted the target in all of CaMwereF19,L32,M36,M51,andM72,allhydrophobic them,asdidE14.However,thefrequencyofuseofother sidechainsintheNdomain(bluelettersinFigure5A).In FLMM residues(L32,M51,andM71)waslowerandwas N thecommonparlanceofCaM-peptideinteractions,F880 dispersed amongotherhydrophobic N-domainresidues andT886qualifyaspeptideanchorsinthehydrophobic (L18,M72,M36,F68,andL39)thatcontactedthetarget clefts of the C and N domains, respectively; however, inasmanyormorestructures.Anoverlayof17Ndomains the anchors are usually both hydrophobic. Alignment of aligned according to Ca atoms of the FLMM tetrad N theCaMsequencebyitscalcium-bindingsites(Figure5A) (Figure6D)showsthatthepocketformedisverysimilar illustrated that a tetrad of the CaM residues contacting inallstructures. F880andT886residueswereasetofidenticalsidechains AcomparisonofsidechainorientationsofeachFLMM (FLMM) in corresponding positions: F19/F92, L32/L105, residueinthese17structuresisshowninFigure6E.Each M51/M124,andM71/M144(boxed,Figure5A). FLMM residue was aligned with the corresponding resi- The domains of CaM in complex with NR1C1p were due in 2HQW; rmsds ranged from 0.2 to >1.6 A˚. All alignedbyminimizingthedistancebetweentheCaatoms FLMMresidues,exceptF92,hadsidechainorientations oftheFLMMtetradresiduesineachdomain;theirback- that deviated by <1.0 A˚ in most structures; the smallest bone structures were closely aligned (Figure 5B). The deviations were observed for M residues. For residues rmsdfortheCaatomsoftheFLMMtetradineachdomain F19,L32, and F92,deviations ranged from1.2 to 1.4 A˚. was0.208A˚,andthisvaluewas0.573A˚ foracomparison In these, F19 and F92 were rotated by (cid:4)90(cid:3) relative to ofthewholedomain.Thus,theFLMMresiduesineachdo- the orientation observed in 2HQW (Figures 6F and 6H), 1606 Structure15,1603–1617,December2007ª2007ElsevierLtdAllrightsreserved Structure CalmodulinBoundtoNMDARNR1C1Region Figure4. DistributionofCaMN-andC-DomainContactsintheCaM-NR1C1pComplex (A)N-domainresidues%4.5A˚ ofNR1C1pshownassticks;17contactsweremadewithNR1residues875–885(gray),and19contactsweremade withresidues885–896(black). (B)SequencemapofCaMresidues%4.5A˚ofNR1C1p.ResiduesinNR1C1pthatmakethehighestnumberofcontactsexclusivelywiththeCdomain (F880)andtheNdomain(T886)areboxed;theERretentionsignalisunderlined. (C)C-domainresidues%4.5A˚ofNR1C1pshownassticks;27contactsweremadewithresidues875–885,and7contactsweremadewithresidues 885–896.Ca2+ionsandbindingsites(yellowin[A]and[C])aredesignatedI,II,III,andIV. ThefigurewasmadewithMacPymol. Structure15,1603–1617,December2007ª2007ElsevierLtdAllrightsreserved 1607 Structure CalmodulinBoundtoNMDARNR1C1Region Figure5. ComparisonofFLMMTetrads inNandCDomainsofCaM (A)SequencealignmentoftheCaMNdomain (1–75) and C domain (76–148). Blue boxes highlightF19,L32,M51,andM71;redboxes indicateF92,L105,M124,andM144.Yellow boxes indicate calcium-binding sites. Resi- duescontactingK875arepurpleandunder- lined. (B)TheCaMNdomain(blue;residues8–73) andCdomain(red;residues81–146)aligned accordingtoCaatoms(green)oftheirFLMM tetrad residues. Ca2+ ions and binding sites areyellow. (C)ComparisonofFLMMresiduesidechains (sticks) after alignment of Ca atoms (green spheres). (D)ElectrondensityofFLMM andF880shown C atacontourlevelof1.0s. ThefigurewasmadewithMacPymol. andLvariedmostattheCgandCdatoms(seeFigure6G; theFLMM tetrad.Astructuralalignment ofthesecom- C Table S1, see the Supplemental Data available withthis plexes according to the Ca atoms of the FLMM (Fig- C articleonline). ure7B)revealedthattheorientationsoftheresiduecon- tacting the majority of these FLMM residues in all 13 C IdentifyingTargetResiduesthatContact structureswerewellconserved. theFLMMTetradsofCaM Theresidueineachpeptidethatcontactedthehighest An analysis of the chemical characteristics of the target numberoftheFLMM residuesisboxedinblueinFigures N residues that contact FLMM and FLMM revealed that 7Aand8.InthecaseofCaMboundtoCaMKIIa(1CDM), N C these tetrads were not used identically by the targets. smMLCK(2BBM),andhRyR1(2BCX),morethanoneres- Figure7Ashowssequencesofthepeptidein13compact idue contacted an equal number of FLMM residues. In N CaM-targetcomplexes.Thesewerealignedaccordingto each structure, the residue that contacted the majority thepeptideresidue(redbox)thatcontacted thehighest ofFLMM residueswasalsotheonethathadthehighest N numberofFLMM residues.In11of13complexes,this number of contacts with all N-domain residues of CaM, C residue also made the highest number of contacts with withthe exception of the CaM-CaMKI structure (1MXE), allC-domainresiduesofCaM(Figure8,redbars).Intwo in which R317 contacted one more residue than M316. cases, there were 2 residues (Y1627 and F1628 of the However, unlike contacts in the C domain, there were Ca 1.2 channel in 2BE6, and W3620 and L3623 of otherresiduesinthetargetthathadthesamenumberof v hRyR1in2BCX)thateachcontacted3FLMM residues; contacts as these. For example, in the CaM-CaMKIIa C F1628in2BE6andW3620in2BCXhadthehighestnum- structure(1CDM),R297,G301,L304,T305,andA309all berofcontactswithallC-domainresiduesofCaM.In12of contactedthesamenumberofN-domainresidues(4)in these13structures,theresiduethatcontactedthehighest CaM;however,ofthese,onlyT305andA309contacted numberofFLMM residueshadalargearomaticmoiety themajorityoftheFLMM residues. C N (7 F, 5 W, 1 Y) and two (CaMKII [1CDM] and hRyR1 A large variation was observed in the size, chemical [2BCX]) had a leucine residue in the cavity defined by characteristics, and spacing of the residues that 1608 Structure15,1603–1617,December2007ª2007ElsevierLtdAllrightsreserved Structure CalmodulinBoundtoNMDARNR1C1Region Figure 6. Statistical Analysis of CaM- TargetInterfaces (A–D)Histogramsshowingresiduesinthe(A)C domain(residues84–146)or(C)Ndomain(res- idues5–72)ofCaM%4.5A˚fromaboundpep- tide ordrugin more than 11 of17 compact CaM-target structures. Bars for residues in theFLMMtetradsareblack;othersaregray. Only residues defined in all 17 structures wereanalyzed.Alignmentof13CaM-peptide complexesbytheCaatomsoftheir(B)FLMMC (red)or(D)FLMMN(blue)residues;theirside chainsareshownassticks.Thedomainsur- faceof2HQWiscoloredpink(Cdomain)or lightblue(Ndomain). (E) A histogram of rmsds for residue side chainsinFLMM andFLMM in16CaM-target N C structurescomparedtothecorrespondingres- iduein2HQW(seeTableS3). (F–H)Binsrepresentincrementsof0.2A˚.Com- parisonofsidechainorientationsofrepresen- tative FLMM residues with high rmsds from 2HQW(black):(F)F19in2BCX(blue),1SY9(or- ange), and 1MXE (green); (G) L32 in 2BCX (blue),2BE6(red),1CKK(orange),and1CDL (green); and (H) F92 in 1NIW (orange) and 1MXE(green). ThefigurewasmadewithMacPymol. contactedthemajorityoftheFLMM residuesrelativeto andmakesthehighestnumberofcontactswiththeNdo- N the primary anchor residue at the reference position of mainofCaM,appearstocontactonlytherimofthecavity ‘‘1’’(Yapetal.,2000).In11ofthesequences,theresidue definedbytheFLMM tetrad(Figure7D). N contactingFLMM wasahydrophobicaminoacid,butits ToexplorethegeneralavailabilityoftheFLMMtetrads N positionvariedfrom10,11,14,16,or17.Thestructuresof tobindingofahydrophobicmoietythatisnotrestricted CaM-NR1C1p(2HQW),CaM-MARCKS(1IWQ),andCaM- by the orientation and chemical linkage of residues in a CaMKIIa(1CDM)wereunusualinthattheresiduecontact- target peptide, this analysis of the FLMM tetrads was ingthemajorityoftheFLMM residueswaspolar(Seror focused to include those in four CaM-drug compact N Thr)andatposition7.Astructuralalignmentofthesecom- complexes. Three of these have TFP (Trifluoperazine; plexes according to the Ca atoms of the FLMM 10-[3-(4-methyl-piperazin-1-yl)-propyl]-2-trifluoromethyl-10- N (Figure7C)revealedamuchlargervariationintheposition phenothiazine)boundin3CaM:drugratios(1:1,1:2,1:4), of the target residue in the FLMM cavity than was ob- andthefourthhasDPD(N-[3,3,-diphenylpropyl]-N0-[1-R- N served for the FLMM cavity. In some structures (i.e., (3,4-BIS-butoxylphenyl)-ethyl]-propylenediamine) bound C complexes withpeptidesfromMARCKS,CaMKIIa,NR1 in a 1:2 ratio of CaM:drug (1QIV.pdb). In the structures C1,eNOS,andMyosinVI),theFLMM cavitywasempty witha1:1ratioofCaM:TFP(1CTR.pdb)andwitha1:2ratio N or only partially occupied, as illustrated in Figure 7D for (1A29.pdb),onlytheFLMM tetradwasoccupied,andin C theCaM-NR1C1pstructure.TheC-domainprimarycon- the structure with a 1:4 ratio of CaM:TFP (1LIN.pdb) tactresidueofthepeptideinthisstructure(F880)wasob- bothtetradswereoccupiedbyTFP.Astructuralalignment servedtofilltheFLMM cavityofCaM.However,T886, ofthetwodomainsof1LIN.pdbaccordingtothepositions C which contacts the majority (3) of the FLMM residues oftheCaatomsoftheFLMMtetradsillustratedthatTFP N Structure15,1603–1617,December2007ª2007ElsevierLtdAllrightsreserved 1609 Structure CalmodulinBoundtoNMDARNR1C1Region Figure7. DistributionandOrientationofTargetResiduesContactingCaMinCompactCaM-PeptideComplexes (A)Sequencesof13peptidesalignedbytheresidue(redbox)thatcontactedthemajorityofFLMMCresidues;thepeptideresiduethatcontactedthe majorityofFLMMNresiduesisboxedinblue.Thenumbersabovethesequencesdenotethespacingbetweenthese2residues.Tenpeptidesbindto CaMinanantiparallelorientation;sequencesareshownbyusingthestandardconvention(theN-terminalresidueisleftmost).Threepeptidesnoted byanasterisk(*)andlistedlastbindtoCaMinaparallelorientation;theirsequencesareshowninreverse. (BandC)Alignmentof13CaM-peptidecomplexesbytheCaatomsoftheirFLMMresidues.Theseresiduesin2HQWareshownasredspheres;the sidechainsoftheprimarycontactresidueofthetargetareshownasblacksticks.Thedomainsurfaceof2HQWiscoloredpink(Cdomain)orlightblue (Ndomain). (D)FLMMpocketoccupancyin2HQW.FLMM (red)andFLMM (blue)residuesofCaM,aswellasF880(black),T886(black),andF891(gray)of C N NR1C1pin2HQWareshownassticks. (E)DrugoccupancyoftheCaMdomains.AlignmentoftheNdomain(blue,residues8–73)andCdomain(red,residues81–146)ofCaMina1:2 DPD:CaM(1QIV.pdb)bytheCaatomsoftheirFLMMresidues.DPDboundtotheCaMNdomainisblack,andDPDboundtotheCdomainisgreen; thetransparencyofdomainswas0.5. ThefigurewasmadewithMacPymol. wascapableofbindingbothFLMMcavitiesinthesame in the DPD complex versus 0.573 A˚ in the NR1C1p orientation, and that the two domains of CaM bound to complex). TFPhavesimilarstructures(rmsdof0.492A˚).Thissimilar- itybetweendomainswasalsoobservedintheCaM-DPD DISCUSSION structure,inwhichbothFLMMtetradswereoccupiedwith thesamemoietyofaDPDmoleculeinthesameorienta- TheC1regionoftheNR1subunitoftheNMDAreceptor tion(Figure7E).TheNandtheCdomainsofCaMinthis has been shown to regulate receptor trafficking and structure align nearly as well as the domains of CaM decreasePKC-inducedreceptorpotentiation.Thestruc- when bound to NR1C1p (Figure 5B) (rmsd of 0.652 A˚ tureof(Ca2+) -CaMboundtoNR1C1p(2HQW)presented 4 1610 Structure15,1603–1617,December2007ª2007ElsevierLtdAllrightsreserved Structure CalmodulinBoundtoNMDARNR1C1Region Figure8. InterfaceAnalysisof13CaM-PeptideComplexes ResiduesintheNdomain(gray)andCdomain(black)ofCaMwithin4.5A˚ ofapeptideresiduedeterminedwithCSU.Redindicatesthepeptideres- iduecontactingthehighestnumberofC-domainresidues;blueindicatesthepeptideresiduecontactingthehighestnumberofN-domainresidues. heredemonstratesthatC1isa1-7motif,anditindicates tation of NR1C1 mutants that would disrupt association which residues contribute to the interface between with CaM and serve to test the role of CaM in NR1 CaMandNR1C1.Itprovidesafoundationfortheinterpre- trafficking. Structure15,1603–1617,December2007ª2007ElsevierLtdAllrightsreserved 1611 Structure CalmodulinBoundtoNMDARNR1C1Region PhysiologicalSignificanceofthe highlightedagroupof7hydrophobicresidues(4M,2F, CaM-NR1C1pComplex 1L)ineachdomainofCaMthatsurroundthehydrophobic NR1subunitsareexpressedinan(cid:4)10-foldexcessover pocketsinthesestructures.Onthebasisofacomprehen- NR2subunitsinthecell;however,only40%–50%ofthese sive statistical analysis of the contact distances in 17 NR1subunitsreachthecellsurfaceinculturedhippocam- wrap-around CaM-target structures, we have identified pal neurons (Okabe et al., 1999). There are eight splice theFLMM tetradasprongsthatholdahydrophobicres- C variantsofNR1,andthemajorvariantinthebraincontains idueinallcanonicalCaM-targetcomplexesstudiedhere, theC0-C1-C2regions(MoriandMishina,1995);however, whereasthecorrespondingFLMM tetradisnotaswell N thisvarianthasthelowestfractionofcell-surfaceexpres- contacted. sion(Okabeetal.,1999).ERretentionoftheNR1subunit ismediatedbyasequenceofthreecontiguousRresidues ConservationofFLMMTetradsinCaMSequences ontheC1regionandiscontrolledbythephosphorylation GiventheprevalenceoftheFLMMtetradsinthetarget- ofS896aftertheERretentionsignal(Scottetal.,2001). bindingpocketsofCaM,itwasexpectedthattheseresi- Thestructureof(Ca2+) -CaMboundtoNR1C1pshowed dueswouldbeconservedinthesequenceofCaMacross 4 thattheERretentionsignalandS896werenotoccluded species.Comparisonof102CaMsequences(Figure9)re- byCaM.Whilethisstructurealonecannotruleoutthepos- vealedthat4ofthe8FLMMresidues(F19,L32,F92,and sibility ofadirectroleofCaMinERretention, itismore M124) were completely conserved, and 2 (M51, L105) likely that CaM might serve as an indirect modulator by were 99.98% conserved: position 51 was M in all but interacting with a kinase or other protein that has a role two sequences (Calm_Yeast and Calm_KLULA have inERretention. L), and position 105 was L in all but two sequences StudiesofNR1splicevariantscontainingtheC1region (Calm2_PethyhasVandCalm_MouschasW;seeTable showedthatthisregionwasnecessaryandsufficientfor S2forcompleteanalysisandaccessionnumbers).Inthe the formation of discrete subcellular receptor clusters 102 CaM sequences analyzed, this was the only occur- thatareassociatedwiththeplasmamembranewhenex- renceofWatanyposition.Itispossiblethatasequencing pressedinfibroblastcells(Ehlersetal.,1995).Phosphor- ambiguity could account for the two substitutions ylationofS890withintheC1regionbyPKCdisruptsthe observed at this position. Codons for W (UGG) and V receptor-enrichedclusters,resultinginanevendistribu- (GUG) differ by a single base from the sequence for L tionoftheNR1subunit(Tingleyetal.,1997).Thisresidue (UUG),raisingthepossibilitythatL105isalsocompletely hadonly41%solventaccessibilitywhenincomplexwith conserved. (Ca2+) -CaM,anditshydroxylgroupwaswithin4A˚ from Thehydrophobicityoftheremaining2FLMMtetradres- 4 thesulfurgroupsofM36andM51ofCaM.Wehaveshown idues(M71,M144)correspondingtothefourthpositionof that(Ca2+) -CaMbindsNR1C1pwithahighaffinity(K = thetetradinbothdomainsofCaM(Figure5A)washighly 4 D 2nM).Ehlersandcoworkers(Ehlersetal.,1996b)reported conserved among the 102 sequences compared; how- comparableaffinitiesforNR1C1pandalargerfusionpro- ever,these2residuesshowedhighersequencevariation teinofNR1C1p((cid:4)150kDa;K =4nM).Together,these than the other 6 FLMM residues. Position 71 was M in D datasuggestthatCaMcanprotectthisregionfromother 62sequencesandLintheother40.Position144wasM proteinsinthepresenceofcalcium. in67sequences,Vin23,Lin10,andIin2.Thesmaller size of the variant side chains may allow the pocket to CommonFeaturesofHydrophobicCavities accommodatelargeranchorresidues. inCaMDomains Early studiesofCaM-peptide interactions demonstrated ConservationofFLMMTetrads the importance of hydrophobic residues found on both A search of the SWISS-PROT knowledgebase and the thetargetandCaM,particularlytheroleoftryptophanin Protein Data Bank (PDB) for all proteins with identical apeptideandthemethionine‘‘puddles’’oftheCaMhy- spacing of the primary sequence for FLMM (e.g., F- drophobicclefts(O’NeilandDeGrado,1990).Subsequent (x12)-L-(x18)-M-(x19)-M) with PROSITE (Sigrist et al., structural comparisons have concluded that large hy- 2002) on the ExPASy Proteomics Server (http://ca. drophobic residues of the target bind the hydrophobic expasy.org)(Gasteigeretal.,2003)identified361nonre- pocketsofCaM,andthatorientationofbindingisdeter- dundant sequences in these databases (i.e., for se- minedbytheelectrostaticcharacteristicsofthedomains quences found both in the PDB and the SWISS-PROT ofCaM(cf.Bhattacharyaetal.,2004;HoeflichandIkura, knowledgebase, the PDB sequence was omitted; Table 2002;IshidaandVogel,2006;VetterandLeclerc,2003). S3).Ofthese,132weresequencesofCaMwith2listings TherolesofindividualMetresiduesonCaMinenzymeac- peraccessionnumber,1foreachdomain.Thereweresix tivation have been investigated via mutagenesis (Chin structures available in the PDB of non-CaM sequences etal.,1997).Dynamicssimulationsfurthersupportthethe- that contained an identical FLMM primary sequence: sisthatasetofconservedmethioninesineachdomainof CaM-like protein 3(1GGZ-A), the C domain of CaM-like CaMareflexibleandallowforaccommodationofvariable protein 5 (2B1U-A), the N domain of centrin (caltractin; peptideresidues(Fiorinetal.,2006).Astructuraloverlayof 2AMI-A), cytochrome P450 (2IJ5-A), aspartyl-rTNA syn- the domains of CaM in seven wrap-around CaM-target thase(1C0A-A),andtheMchainofthephotosyntheticre- structures available in the PDB (Ishida and Vogel, 2006) action center (1AIJ-M). Alignment of Ca atoms of the 1612 Structure15,1603–1617,December2007ª2007ElsevierLtdAllrightsreserved
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