Role of Conserved Salt Bridges in Homeodomain Stability and DNA Binding* S

34 Pages·2009·8.42 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.284,NO.35,pp.23765–23779,August28,2009
©2009byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
Role of Conserved Salt Bridges in Homeodomain Stability
and DNA Binding*□S
Receivedforpublication,April23,2009,andinrevisedform,June25,2009 Published,JBCPapersinPress,June26,2009,DOI10.1074/jbc.M109.012054
MarioTorrado‡1,JuliaRevuelta‡,CarlosGonzalez§,FranciscoCorzana¶2,AgathaBastida‡,andJuanLuisAsensio‡3
Fromthe‡DepartamentodeQuímicaOrga´nicaBiolo´gica,InstitutodeQuímicaOrga´nicaGeneral,ConsejoSuperiorde
InvestigacionesCientíficas,28006Madrid,§GrupodeRMN,InstitutodeQuímica-Física“Rocasolano,”ConsejoSuperiorde
InvestigacionesCientíficas,28006Madrid,andthe¶DepartamentodeQuímica,UniversidaddelaRioja,UnidadAsociadaConsejo
SuperiordeInvestigacionesCientíficas,E-26006Logron˜o,Spain
The sequence information available for homeodomains Presently,thelargestsearchablecollectionofinformationfor
reveals that salt bridges connecting pairs 19/30, 31/42, and the homeodomain protein family is the Homeodomain
17/52arefrequent,whereasaliphaticresiduesatthesesitesare ResourceDataBase(5–7).Itcontainsaround1056full-length
rareandmainlyrestrictedtoproteinsfromhomeotherms.We proteinsequencesisolatedfrom112differentspecies.Inspec-
haveanalyzedtheinfluenceofsaltandhydrophobicbridgesat tionofthesedatashowsthathelicesI/II,I/III,andII/IIIcanbe
these sites on the stability and DNA binding properties of connected by three salt bridges involving pairs 19/30, 31/42,
humanHesx-1homeodomain.Regardingtheproteinstability, and17/52,respectively,whichinadditioncanexhibitdifferent
ouranalysisshowsthathydrophobicsidechainsareclearlypre- polarities. In contrast, hydrophobic bridges at these sites are
ferredatpositions19/30and31/42.Thisstabilizinginfluence present only in a minor fraction of sequences (and mainly
results from the more favorable packing of the aliphatic side restricted to homeodomains from warm-blooded animals).
chainswiththeproteincore,asillustratedbythethree-dimen- Thispredominanceofsaltversushydrophobicbridgessuggests
sional solution structure of a thermostable variant, herein arolefortheformerinhomeodomainfunctionand/orstability.
reported.Incontrastonlypolarsidechainsseemtobetolerated Hereweanalyzetherelativeeffectofsaltandhydrophobic
at positions 17/52. Interestingly, despite the significant influ- bridgesonthehomeodomainstabilityandDNAbindingprop-
enceofpairs19/30and31/42onthestabilityofthehomeodo- erties,employingthehumanHesx-1DNAbindingdomainas
main,theireffectonDNAbindingrangesfrommodesttoneg- modelsystem.Ourapproachinvolvestheextensiveuseofsite-
ligible. The observed lack of correlation between binding directed mutagenesis experiments together with CD, NMR,
strengthandconformationalstabilityintheanalyzedvariants and isothermal titration microcalorimetry (ITC)4 measure-
suggests that salt/hydrophobic bridges at these specific posi- ments. Thus, as a first step, the hydrophobic pair Val19/Ile30,
tionsmighthavebeenemployedbyevolutiontoindependently present in the wild-type polypeptide, was replaced by salt
modulatebothproperties. bridgesofdifferentpolarities,eitherisolatedornetworked.A
previous statistical analysis of the information stored in the
homeodomainresourcedatabase(8,9)revealedanintriguing
Homeodomainproteinsaretranscriptionfactorspresentin correlationbetweenthenatureofpair19/30andtheloopresi-
alleukaryotesandplaykeyrolesincellulardifferentiationdur- due26(prolineorabranchedaliphaticaminoacidinallcases).
ingdevelopment(1,2).Thehomeodomainencodesa60-resi- Consideringthis,bothsaltbridgesandaliphaticresiduescon-
dueDNAbindingdomaincomposedofadisorderedN-termi- necting19/30werealsoconsideredinthecontextofeitherpro-
nalarm,threehelicalsegments(hereinreferredasI,II,andIII) lineorleucinein26.Finally,conservedsaltbridges31/42and
connectedbyshortloops,andadisorderedC-terminalregion. 17/52weresubstitutedbyhydrophobicpairsobservedinother
Thesecondaryandtertiarystructuresarestabilizedbythefor- naturalsequences.
mationofawelldefinedhydrophobiccore,whichresultsfrom Theworkreportedhereimpliedtheproductionof33home-
thepackingofthethreehelices(3,4). odomainvariants,includingsingle,double,triple,andquadru-
ple mutations. All of them were subjected to careful thermal
*This work was supported by Spanish “Plan Nacional” (Ministerio de and chemical denaturation experiments, employing circular
Ciencia y Technologia) Research Grants CTQ2007-67403/BQU and
dichroism.Thisanalysisallowedtheidentificationofathermo-
CTQ2004-04994/BQU.
Theatomiccoordinatesandstructurefactors(code2k40)havebeendepositedin philic variant of the Hesx-1 homeodomain. Its three-dimen-
theProteinDataBank,ResearchCollaboratoryforStructuralBioinformatics, sionalsolutionstructurewasdeterminedbyNMRmethodsto
RutgersUniversity,NewBrunswick,NJ(http://www.rcsb.org/).
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains provideadetailedstructuralframeworkforelucidatingtheori-
supplementalTablesS1andS2andFigs.S1–S13. ginoftheenhancedthermalstability.Inaddition,thepKaval-
1RecipientofaMinisteriodeEducacio´nyCienciaFormacio´ndeProfesorado uesforcarboxylgroupsinsomeselectedvariantsweremeas-
Universitariofellowship.
2RecipientofaRamo´nyCajalcontract.
3Towhomcorrespondenceshouldbeaddressed:Dept.deQuímicaOrga´nica 4The abbreviations used are: ITC, isothermal titration microcalorimetry;
Biolo´gica, Instituto de Química Orga´nica General, Consejo Superior de NOESY,nuclearOverhausereffectspectroscopy;DTT,dithiothreitol;DQF-
InvestigacionesCientíficas,JuandelaCierva3,28006Madrid,Spain.Tel.: COSY, double quantum-filtered correlation spectroscopy; PDB, Protein
34-91-561-8806 (Ext. 329); Fax: [phone]; E-mail: iqoa110@ Data Bank; GdmCl, guanidinium chloride; TOCSY, total correlation
iqog.csic.es. spectroscopy.
AUGUST28,2009•VOLUME284•NUMBER35 JOURNALOFBIOLOGICALCHEMISTRY 23765
This is an Open Access article under the CC BY license.
SaltVersusHydrophobicBridgesinStabilityandDNABinding
uredbyNMRspectroscopytofurthertesttheroleofconserved initialandfinaldenaturantconcentrationswereconfirmedby
saltbridgesonthehomeodomainstability.Asafinalstep,the refractometry.(cid:3)G(thefreeenergyoffolding)andmvalues(the
DNA binding properties of selected mutants, together with dependence of the free energy of folding on the denaturant
their temperature dependence, were analyzed employing concentration) were determined from the chemical denatur-
microcalorimetry. The results obtained provide new insights ationdataassumingatwo-statetransitionandusingthelinear
into the sequence, structure, stability, and function relation- extrapolation method (10). Nonlinear regression calculations
shipswithinthisimportantfamilyofDNA-bindingproteins. were performed employing Origin 5.0. The errors in the (cid:3)G
values derived from the fitting procedure were in all cases
EXPERIMENTALPROCEDURES
smallerthan0.1kcal/mol.
Site-directedMutagenesisofHesx-1—Mutationswereincor- Thermal unfolding transitions were measured employing
poratedintothewild-typeHesx-1homeoboxlocatedonplas- 1-cm path length quartz cells from samples containing 3 (cid:3)M
midpT7-7usingtheQuikChangemutagenesiskit(Stratagene, proteinin10mMphosphate,0.5mMDTT,pH6,andfivedif-
La Jolla, CA) and the appropriate mutagenic primers. The ferent NaCl concentrations (0, 0.1, 0.4, 1.0, and 2.0 M). The
mutant plasmids were transformed into CaCl2-competent transitionsweremonitoredbythedecreaseoftheCDsignalat
Escherichia coli DH5(cid:1)cells. The sequences of all constructs
222nmusinga2-nmbandwidth.Heatingrateswere20°C/h.
wereconfirmedbyDNAsequencing.
Transitions were evaluated using a nonlinear least squares fit
Protein Expression and Purification—Transformed E. coli
assumingatwo-statemodelwithslopingpre-andpost-transi-
BL21(DE3) cells were grown to A600 (cid:1) 0.6 at 37°C in LB tional base lines. (cid:3)G values at a common temperature (55 or
medium. Then cultures were induced with 0.5 mM isopropyl 77°C depending on the particular set of protein variants, see
1-thio-(cid:2)-D-galactopyranoside. Induction times and tempera- Table1)werederivedfromthethermaldenaturationdata.In
tureswereoptimizedforeverymutantbeingtypically13–24h thisprocedure,aconstantvalueof(cid:4)0.7kcal/(mol(cid:2)K)wasused
at22–28°C.Cellswereharvestedandlysedfollowedbyclarifi- for the change in heat capacity upon folding ((cid:3)Cp). It repre-
cationandremovalofnucleicacidsbypolyethyleneiminepre-
sentstheaverageofallthevaluesfoundintheanalysesofthe
cipitation.Thenucleicacid-freeextractsweredialyzedagainst
individualthermaltransitions.Inaddition,itisconsistentwith
50mMsodiumphosphatebuffer,pH7.5,2mM(cid:2)-mercaptoeth-
the denaturant m values measured for the different protein
anol(BufferA),andthenloadedontoBio-RexA-70(Bio-Rad)
variantsandalsowiththedecreaseintheaccessiblesurfacearea
columnequilibratedinthesamebuffer.Thecolumnwasexten-
estimated for the homeodomain upon folding (11). The fact
sively washed with Buffer A, and then the protein was eluted
that m values exhibit very low variations for the different
with a linear gradient of 0–300 mM NaCl. As the next step, mutants (in all cases it is in the 0.8–1 kcal/(mol(cid:2)M) range for
ammonium sulfate precipitation was employed to further
denaturation experiments with urea and in the 1.4–1.7 kcal/
purify and concentrate the homeodomain variants. Ammo-
(mol(cid:2)M) range for those with guanidinium chloride) supports
nium sulfate was increased stepwise from 45% saturation to
theemploymentofacommon(cid:3)Cpforallofthem(11).Com-
100%,andtheprecipitatedproteinwasseparatedbycentrifu-
gation(20minat12,000(cid:2)g)ateverystage.Theprecipitated parable(cid:3)CpvalueshavebeendescribedfortheEngrailed((cid:4)0.7
kcal/(mol(cid:2)K))(9)andVndK2((cid:4)0.52kcal/(mol(cid:2)K))(12)home-
protein obtained in the last step (100% saturation of ammo-
odomains.Inaddition,ithastobeconsideredthatmostvari-
nium sulfate) was collected, resuspended in 5 ml of 20 mM
sodiumphosphatebuffer,100mMNaCl,2mM(cid:2)-mercaptoeth- ants showed denaturation midpoints relatively close to 55 or
77°C (depending on the set of mutants), and therefore large
anol, pH 7.0 (Buffer B), and loaded onto FPLC Superdex-75
extrapolationswerenotnecessaryfordetermining(cid:3)Gatthese
HiLoad 26/60 (Amersham Biosciences) column previously
temperatures.
equilibrated in the same buffer. The protein was then eluted
The standard errors for T and (cid:3)H determined from the
employingaflowrateof2.5ml/min.Thepurehomeodomain m
analysis of the individual melting profiles were typically not
fractions were pooled and desalted by dialysis against 2 mM
sodiumphosphate,5mMNaCl,2mM(cid:2)-mercaptoethanol,pH higherthan0.1°Cand1kcal/mol,respectively.Theaccuracyin
6.0, and then lyophilized. At the end of the procedure, the the(cid:3)Gvaluesat55or77°CisobviouslycorrelatedwiththeTm.
purity of the proteins was shown to be greater than 95% by Toestimatetheerrorsinthefoldingfreeenergies,theirvalues
SDS-PAGE. The protein concentrations were determined werealsoderivedconsideringavariationof2kcal/molin(cid:3)H
spectrophotometrically. ((cid:3)H (cid:5) 2 kcal/mol), 0.2°C in Tm (Tm (cid:5) 0.2°C), and 0.2 kcal/
CD Spectroscopy—Circular dichroism experiments were (mol K) in (cid:3)Cp ((cid:3)Cp (cid:5) 0.2 kcal/(mol K)). In every case, the
recordedinaJascoJ-810spectropolarimeter,fittedwithaPel- maximumdifferencebetweenthecalculatedfreeenergieswas
tiertemperaturecontrolaccessory. takenastheerror(seesupplementalFig.S1).
GdmCl and urea denaturation data were obtained at 20°C Finally,thecontributionstostabilitystillpresentin2MNaCl
fromsamplescontaining18(cid:3)Mproteinin10mMphosphate, were assumed to reflect primarily packing and hydrophobic
100 mM NaCl, 0.5 mM DTT, pH 6, employing an autotitrator interactions(hereinreferredas(cid:3)G2MNaCl),whereasthosethat
and 1-cm path length quartz cells provided with a magnetic couldbescreenedbyadding2MNaClwereattributedtocou-
stirrer.UreaandGdmClstocksolutionswerealsopreparedin lombic interactions ((cid:3)G ). They were calculated from
coulomb
the same buffer. Chemical denaturations were followed by the difference (cid:3)G (cid:1) (cid:3)G (cid:4) (cid:3)G and
coulomb 0 M NaCl 2 M NaCl
monitoring the CD ellipticity at 222 nm. Data were collected the corresponding errors estimated from E (cid:1)
(cid:3)Gcoulomb
every0.2Mfrom0to9.4Mureaorfrom0to8.5MGdmCl.The ((E(cid:3)G0MNaCl)2(cid:6)(E(cid:3)G2MNaCl)2)1/2.
23766 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER35•AUGUST28,2009
SaltVersusHydrophobicBridgesinStabilityandDNABinding
NMRStructureDeterminationoftheThermostableVariant applied at 5-min intervals, whereas the DNA solution was
R31L/E42L—1H NMR spectra were recorded in 85:15 1H O: stirredataconstantspeedof300rpm.Dilutionheatsofprotein
2
2H Oandin2H OonBrukerAvance800,BrukerAvance600, intoDNAsolutions(whichagreedwiththoseobtainedbyinjec-
2 2
and Varian Unity 500-MHz spectrometers. NMR samples tions of proteins into the same volume of buffer) were sub-
included400–800(cid:3)Mproteinconcentrationin100mMNaCl, tractedfrommeasuredheatsofbinding.Titrationcurveswere
10mMsodiumphosphatebuffer,and2mMDTT,pH6.0.Pro- analyzedwithOrigin,providedwiththeinstrumentbyMicro-
teinassignmentswereobtainedusingasetoftwo-dimensional CalLLC,usingaone-sitebindingmodeltofitthecurves.For
NOESY(13),TOCSY(14)andDQF-COSY(15)experiments. everysingleproteinvariant,thermodynamicparameterswere
TheTOCSY,NOESY,andDQF-COSYexperimentswerecar- derivedfromtwoindependentexperimentsandaveraged.
ried out in the phase-sensitive mode using the TPPI method
(15)forquadraturedetectionintheindirectdimension.Typi- RESULTS
cally, a data matrix of 512*2048 points was used to digitize a
OccurrenceofSaltBridgeswithintheHomeodomainFamily—
spectralwidthof8000–6000Hz.80scanswereusedperincre-
Asafirststep,therelativeoccurrenceofbothsaltbridgesand
mentwitharelaxationdelayof1s.PriortoFouriertransforma-
hydrophobic pairs at positions 19/30, 31/42, and 17/52 was
tion,zerofillingwasusedintheindirectdimensiontoexpand
determinedfromtheinformationstoredintheHomeodomain
the data to 1K*2K. Base-line correction was applied in both
Resource Data Base (1056 nonredundant full-length and
dimensions.TheTOCSYspectrawereacquiredusing60msof
domainsequences).Forouranalysis,adatasetof754proteins,
isotropic mixing period. The NOESY experiments were per-
including the canonical 60-residue homeodomain sequence
formedwithmixingtimesfrom50to200ms.
with no insertions or deletions, was considered. The results
Upper limits for proton-proton distances were obtained
obtainedarerepresentedschematicallyinFig.1,a–c.Itcanbe
fromNOESYcross-peakintensitiesatthreemixingtimes,50,
observed that positions 19/30 are connected by salt bridge
150, and 200 ms. Cross-peaks were classified as strong,
interactions of both polarities in 52.5% of the cases (396
medium, and weak corresponding to upper limits of 2.5, 3.5,
sequences)withaclearpreferenceforthosecombinationswith
and5.0Å.Thelowerlimitforproton/protondistanceswasset
apositivelychargedresiduein30(Fig.1a).Incontrast,aliphatic
asthesumofthevanderWaalsradiioftheprotons.Distance
residuesatthesesitesarerelativelyrare(2.5%).
geometry calculations were performed on a Silicon Graphics
Insomecases,residues19/30canestablishacooperativenet-
O2 computer using the program DYANA (16). A set of 703
workwithpositions15/37,asthatobservedinthex-raystruc-
constraintswasusedinthefinalroundofcalculations.
tureoftheEngrailedhomeodomain(9,20).Anequivalentpat-
The30bestDYANAstructuresintermsoftargetfunction
ternofinteractionsinvolvingpairs19/30and15/37wouldbe
weresubmittedtoasimulatedannealingprotocol(17)withthe
feasibleinonly2.7%ofthesequencesstoredintheHomeodo-
AMBER5.0packageandtheparametersdescribedbyKollman
mainResourceDataBank(seeFig.1b).Infact,analternative
and co-workers (18). To prevent nonrealistic interactions
network involving the triad 19/30/33 (typically Glu19/Arg30/
betweendisorderedregionsoftheproteinandthestructured
Glu33)seemstobemuchmorefrequentwithinthisfamilyof
helicalcore,explicitTIP3P(18)watermoleculesandperiodic
proteins.
boundaryconditionswereemployedinthesecalculations(19).
From a statistical analysis of homeodomain sequences,
pK Measurements—NMR experiments were performed at
a
5°ConaBrukerAvance800spectrometerin85:151H O:2H O. Clarke(8)identifiedadominatingpatternofpairwiseco-vari-
2 2
NMRsampleswerepreparedin100mMNaCl,10mMsodium ation centered on residue 26. Using the co-varying network,
phosphate,and2mMDTTat400–600(cid:3)Mproteinconcentra- homeodomains were divided into two classes. One class has
tions. Resonances for all the glutamate and aspartate side branchedaliphaticresiduesatposition26,whereasthesecond
chains were assigned employing a set of two-dimensional containsproline.Previousanalysisofareduceddatasetofrep-
NOESY(13),TOCSY(14),andDQF-COSY(15)experiments. resentativehumanhomeodomainsequences(9)foundthatthe
Changesinchemicalshiftfortheside-chainH(cid:4)/H(cid:2)protonsin branchedaliphaticsubgroupusuallyhasasaltbridgeconnect-
glutamatesandH(cid:2)protonsinaspartatesweremonitoredasa ing residues 19 and 30 (92% of cases). Strikingly, none of the
function of pH. The pK values were obtained from nonlinear Pro26subgroup,withmorethan60members,hadthispotential
least squares fitting employing the Henderson-Hasselbach interaction.Basedonthisobservation,itwassuggestedthatsalt
equationwithHillcoefficientssetto1. bridge19/30inpresenceofLeu26couldcontributetothecon-
DNABindingExperiments—Bindingstudieswereperformed formationalvariabilityoftheloopbetweenhelicesIandII.This
at15,25,and35°C,in150mMNaCl,20mMphosphate,5mM couldaffecttheabilityofthehomeodomainforinducedfiton
MgCl ,pH6.0,usingaVP-ITCtitrationcalorimeter(MicroCal, bindingtoDNA.
2
LLC)withareactioncellvolumeof1.467ml.Boththeprotein Our analysis of an extended data set with 754 sequences
and duplex DNA (5(cid:7)-GTCTAATTGACGCG-3(cid:7) and its com- revealsaslightlymorecomplexsituation(Fig.1c).Forhome-
plementary5(cid:7)-CGCGTCAATTAGAC-3(cid:7))solutionsweredia- odomainswithasaltbridgeconnecting19/30,26caneitherbe
lyzed against the same buffer prior to ITC experiments to a branched aliphatic residue (usually leucine) or proline,
ensurechemicalequilibration.Typically,4.0–14.0(cid:3)Mduplex dependingonthesaltbridgepolarity.Ifthepositivechargeisat
DNAinthereactioncellwastitratedwitha100–200(cid:3)Msolu- 30,26isabranchedaliphaticaminoacidin(cid:8)96%ofthecases.
tion of the different Hesx-1 variants contained in a 300-(cid:3)l Ontheotherhand,ifposition30isnegativelycharged,then26
syringe. At least 30 consecutive injections of 5–10 (cid:3)l were canbeeitherleucineorproline,althoughleucineisclearlypre-
AUGUST28,2009•VOLUME284•NUMBER35 JOURNALOFBIOLOGICALCHEMISTRY 23767
SaltVersusHydrophobicBridgesinStabilityandDNABinding
FIGURE1.a,representationoftheEngrailed(upperpanel)andHesx-1(middleandbottompanels)homeodomainstructures.Saltbridgeinteractions,conserved
withinthisfamilyofproteins,connectingpositions19/30(upperpanel),31/42(middlepanel),and17/52(bottompanel)areindicated.Inaddition,frequencies
forsaltversushydrophobicbridgesatthesesites,withinadatasetof754homeodomainsequences,areshownontheright.Symbolsh,(cid:6),and(cid:4)standfor
aliphatic(Val,Leu,andIle),positively(His,Arg,andLys),andnegatively(GluandAsp)chargedresidues,respectively.b,schematicrepresentationofthesalt
bridgenetworksinvolvingresidues19/30mostcommonlyfoundinhomeodomains.Populationsforeachpatternofinteractionsarerepresentedontheright.
c,observedcorrelationbetweenpositions19/30andtheloopresidue26inhomeodomains(hstandsforaliphaticandPstandsforproline).
ferred. Interestingly, if residues 19 and 30 are aliphatic, 26 is werecollectedatfivedifferentNaClconcentrations(0,0.1,0.4,
alwaysproline(seeFig.1c). 1.0, and 2.0 M), and the dependence of (cid:3)G with the ionic
Regarding positions 31/42, they form a salt bridge in the strengthatacommontemperature(55or77°C,seeTable1)
majority of homeodomains (61.7%). For a minor fraction of wasanalyzed.
sequences, an aliphatic residue can be found either in 31 Typicalthermalandchemicaldenaturationprofilescollected
(14.7%) or 42 (3.8%). Finally, both are aliphatic in just five forHesx-1mutantsareshowninFig.2aandsupplementalFigs.
sequences(0.7%)ofourdataset(Fig.1a). S3 and S4. It can be observed that the obtained variants are
Themostconservedsaltbridgeinteractionwithinthehome- strongly stabilized by increasing NaCl concentrations. More-
odomainfamilyisthatconnectingpositions17/52,presentin over, in all cases, (cid:3)G exhibits a linear dependence with the
75.1% of the sequences. Hydrophobic residues at either posi- square root of the ionic strength suggesting that the electro-
tion,17or52,canbefoundin0.8and0.1%ofthecases,respec- static screening of unfavorable interactions is the dominant
tively.However,ahydrophobicpairatthissiteisnotpresentin mechanismfortheobservedsaltstabilization(21).
anysingleproteinwithinourdataset(Fig.1a).Aselectionof Free energies derived at 0 and 2 M NaCl are referred as
representative homeodomain structures with salt bridges (cid:3)G and(cid:3)G ,respectively.Followingthemeth-
0MNaCl 2MNaCl
19/30,31/42,and17/52isshowninsupplementalFig.S2. odology described by Schmid and co-workers (22–25), the
SaltVersusHydrophobicBridgesasDeterminantsofHome- contributions to stability still present in 2 M NaCl were
odomainStability,MethodologicalAspects—Toanalyzetherel- assumedtoreflectprimarilypackingandhydrophobicinter-
ative efficacy of salt versus hydrophobic bridges at positions actions. Other effects, such as desolvation penalties, the
19/30, 31/42, and 17/52 in the stabilization of the homeodo- intrinsichelixpropensitiesofthedifferentresidues,ortheir
mainfold,severalsingle,double,triple,andquadruplemutants conformational entropies, might also contribute to this
ofHesx-1homeodomainwereproduced.Theobtainedvariants term.Ontheotherhand,thosecontributionsthatcouldbe
(1-33inTable1)weresubjectedtobothheatandchemically screened by adding 2 M NaCl were attributed to coulombic
induceddenaturationexperiments. interactions ((cid:3)G ). Throughout this paper, residues
coulomb
To gain further insights into the different contributions to relevant for the discussion in the different protein variants
theobservedchangesinstability,thermaldenaturationprofiles areindicatedinparentheses.
23768 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER35•AUGUST28,2009
SaltVersusHydrophobicBridgesinStabilityandDNABinding
TABLE1
Stabilitydatameasuredforwild-typeHesx-1andvariants1–33fromchemical(left)andthermal(right)denaturationexperiments(employing
ureaorguanidiniumchlorideasindicated)
Mutantsarenumberedaccordingtotheirthermalstabilityat0MNaCl.Forallvariantsaprolineresidueispresentattheloopposition26unlessexplicitlystated(in
parentheses,variants1and17).
a m(kcal/(molM))slopeof(cid:3)Gversusdenaturantconcentrationplots.
bc(cid:3)TGmiisstthheecmhiadnpgoeinintoGfibthbestfhreeermenaelrugnyfoofldfoinldgitnrganatsi5ti5oonr.V77al°uCes(amsienadsuicraetdeda)t;0(cid:3)aGnadta0tM2MNaNCalCisltchoentcoetnatlrcahtiaonngaer,e(cid:3)sGhoatw2nM. NaClrepresentsthenonpolarcontribution,andthe
differencebetween0and2MNaClrepresentstheCoulombiccontributionto(cid:3)G.
dm1(kcal/(molM1/2))slopeof(cid:3)Gversus[NaCl]1/2determinedasdescribedbyRiosandPlaxco(21).Thisparameterisproportionaltothe(cid:3)Gcoulombterm.
AUGUST28,2009•VOLUME284•NUMBER35 JOURNALOFBIOLOGICALCHEMISTRY 23769
SaltVersusHydrophobicBridgesinStabilityandDNABinding
phobic core than the charged side
chains. Even variants 11 (Arg19/
Glu30, Glu15/Lys37), 12 (Arg19/
Glu30,Glu15/Arg37),and15(Glu19/
Arg30, Arg15/Glu37), in which a
network of polar interactions
involving positions 15, 19, 30, and
37 (similar to that observed in the
Engrailed homeodomain) might be
feasible, are severely destabilized
with respect to the wild-type
polypeptide,asaresultoftheunfa-
vorable salt-independent contribu-
tionsofthemutations.
Finally,theobtainedresultsindi-
cateaclearpreferencefortheGlu19/
Arg30combination.Indeed,variants
19 (Glu19/Arg30, Val15/Lys37) and
15 (Glu19/Arg30, Arg15/Glu37) are
more stable than 16 (Arg19/Glu30,
Val15/Lys37) and 12 (Arg19/Glu30,
Glu15/Arg37), respectively (see
Table1).Itislikelythatthispartic-
ular distribution of amino acids
allowsabetterpackingoftheside-
FIGURE2.StabilityanalysisofHesx-1mutantswithdifferentcombinationsofaliphaticandchargedside
chainsatpositions19/30and15/37(indicatedforeverymutant).Residuesatthesepositionsinwild-type chain methylene groups with the
Hesx-1areVal19/Ile30andVal15/Lys37.a,chemicaldenaturationcurvesmeasuredforvariants24,22,3,and12 protein hydrophobic core, and/
at20°Cin0.1MNaCl,10mMphosphate,0.5mMDTT,pH6.0.Thecorrespondingthermaldenaturationcurvesin or a more optimized interaction
presenceof0,0.1,0.4,1,and2MNaClarerepresentedinthelowerpanel.Inallcases,saltincreasesthethermal
stability.b,thermodynamicparametersderivedfortheanalyzedvariants.Meltingtemperaturestogetherwith between position 30 and the loop
freeenergyincrements(withrespecttothemoreunstablemutant,2)derivedfromthechemical(at20°C)and connectinghelicesIandII.Interest-
thermal(at55°Cinthepresenceof0and2MNaCl)denaturationexperimentsarerepresented.Differencesin
the(cid:3)G term((cid:3)(cid:3)G )arealsorepresented.Thosevariantswithapotentialnetworkofsaltbridges ingly, the observed preference is
coulomb coulomb
involving19/30and15/37,togetherwiththewild-typeprotein,areschematicallyrepresentedabove. reflected in the relative abundance
of both types of contacts in the
Salt Bridges Versus Hydrophobic Pairs at Positions 19/30— homeodomain resource data base (salt bridges of equivalent
First,theinfluenceofpair19/30onthestabilityofHesx-1was polaritytothosepresentinmutants19and16arepresentin
analyzed.Itiswellestablishedthatformationofextensivecoop- 33.8 and 18.7% of the sequences, respectively, see Fig. 1a). In
erative networks can greatly enhance the stabilizing effect of conclusion,simplehydrophobicinteractionsestablishedbyali-
saltbridgesinproteins(26).Pair19/30hasbeenshowntobe phaticresiduesatpositions19and30provideamoreeffective
involvedinapolarnetworkwithresidues15/37intheEngrailed
stabilizationoftheHesx-1foldthansaltbridgesofanypolarity
homeodomain (Fig. 1b). Taking this into account, variants,
eitherisolatedornetworked.
including different combinations of charged/aliphatic side
Interaction between Pair 19/30 and the Loop Residue 26,
chainsatthesefourpositions,wereconsidered.Theobtained
Influence on Homeodomain Stability—As indicated under
results are represented in Table 1 and summarized in Fig. 2b
“OccurrenceofSaltBridgeswithintheHomeodomainFamily,”
andsupplementalFigs.S4–S7.
thehomeodomainresourcedatabaserevealsanintriguingcor-
Itcanbeobservedthatthesingleordoublereplacementof
relationbetweenpair19/30andresidue26,locatedontheloop
residues Val19/Ile30 (present in the wild-type polypeptide) by
betweenhelicesIandII.
chargedsidechainstobuildsaltbridgesofdifferentpolarities
Wild-typeHesx-1includesaprolineatposition26,andres-
results,inallcases,inacleardestabilizationofthehomeodo-
idues19/30arebothaliphatic(Val19/Ile30,seeFig.3).Thispair
mainfold.Interestingly,thiseffectseemsrelativelyindepend-
wasreplacedbyasaltbridgeGlu19/Arg30inthepreviouslyana-
entonthecontextprovidedbypair15/37.Thesaltdependence
exhibitedby(cid:3)Gat55°Cprovidesapossibleexplanationforthis lyzedmutants19and15,resultinginaratherunusualcombi-
behavior. Thus, although the unfavorable influences of the nation(Glu19/Pro26/Arg30).Toassesstherelativeeffectofali-
mutations are, in most cases, slightly attenuated at 2 M NaCl phaticversussaltbridgesconnectingsites19/30ontheprotein
(indicating a less optimized electrostatic balance in the stability,inthecontextofbothprolineorleucineatposition26,
mutants), to a large extent they are maintained at such ionic two additional protein variants were produced and analyzed
strength(see(cid:3)G ,(cid:3)G ,and(cid:3)G termsin (Fig.3a).First,thetriplemutant17(Glu19/Leu26/Arg30),which
0MNaCl 2MNaCl coulomb
Table 1 and Fig. 2b), suggesting that Val19 and Ile30 establish contains the triad more frequently found in homeodomains,
moreoptimizedpackinginteractionswiththeproteinhydro- was obtained. Second, Pro26 was replaced by a leucine in the
23770 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER35•AUGUST28,2009
SaltVersusHydrophobicBridgesinStabilityandDNABinding
FIGURE3.a,schematicrepresentationofwild-typeHesx-1togetherwithvariants1,19,and17,characterizedbydifferentnaturalandnon-naturalcombinations
of residues at positions 19, 30, and 26. (cid:3)G values at 20°C measured from chemical denaturation experiments are represented in black. In addition,
(cid:3)G /(cid:3)G /(cid:3)G termsat55°C,derivedfromthermalunfoldingexperiments,areshowningray.Forvariant1,stabilitycouldnotbeanalyzeddue
0MNaCl 2MNaCl coulomb
tostrongaggregationandprecipitationoftheprotein.Freeenergycontributionsaregiveninkcal/mol.b,one-dimensionalNMRspectrameasuredfor
wild-typeHesx-1andvariants17and19.
wild-type protein to produce mutant 1 characterized by the ingintoaccountthatresidue26isnotsolvent-exposedinthe
unnaturaltriadVal19/Leu26/Ile30. native protein (and therefore it is not likely to significantly
Asapreliminaryanalysis,highresolutionone-andtwo-di- affect the protein solubility), this behavior suggests that the
mensionalNMRspectrawerecollectedformutants17(Glu19/ P26Lmutationstronglydestabilizestheprotein.Thisconclu-
Leu26/Arg30) and 19 (Glu19/Pro26/Arg30) and compared with sionmightseemsurprisingconsideringthatitappearsconserv-
that corresponding to the wild-type polypeptide (Fig. 3b). All ative at first glance. However, it would be in agreement with
spectra exhibit sharp signals with a similar dispersion. More- previousstudiesonproteindesign(27–30).Morespecifically,it
over,distinctchemicalshiftsandkeylongrangeNOEs,which has been shown that although some variations in the buried
arehallmarkersofthisfamilyofproteins,couldalsobeidenti- positions of a protein are allowed, there are limits on the
fiedforthetwomutants,whichruleouttheexistenceofmajor sequencesthatresultinstablenative-likefolds(27–30).Taking
structuraldifferencesbetweenthethreehomeodomains.Next, thisintoaccount,specificpackingbetweenresidues19/26/30
both17(Glu19/Leu26/Arg30)and19(Glu19/Pro26/Arg30)were seemscriticalforthestabilityofthehomeodomainfold.
subjected to denaturation studies. The results obtained are Salt Bridges Versus Hydrophobic Pairs at Positions 31/42,
shownschematicallyinFig.3a.ItcanbeobservedthattheP26L Three-dimensional Solution Structure of a Thermostable
substitutionin19(Glu19/Pro26/Arg30),togive17(Glu19/Leu26/ Homeodomain Variant—To determine the influence of pair
Arg30),hasaminoreffectonthehomeodomainstability.Infact, 31/42onthehomeodomainstructureandstability,additional
17(Glu19/Leu26/Arg30)isslightlylessstable,accordingtoboth single and double mutants were prepared and analyzed. The
chemical and thermal denaturation experiments, mainly results obtained are schematically shown in Fig. 4a. It can be
reflectingalessoptimizedelectrostaticbalance(see(cid:3)G observedthatthesinglereplacementE42L(variant28(Arg31/
coulomb
termsinTable1).Aspeculativebutplausibleexplanationfor Leu42))slightlyincreasestheproteinstability(seeTable1and
thisresultisthattheP26Lmutationinducesminorconforma- Fig.4a).Thisresultwasunexpectedgiventheexposedlocation
tionaldifferencesintheloopbetweenhelicesIandII(aflexible of residue 42 and considering that the E42L substitution dis-
regioninhomeodomains),whichinturnmightleadtoapoorer ruptsthesaltbridgepresentinthewild-typepolypeptide(and
electrostatic interaction between charged residues Glu19 and observedinmosthomeodomainstructures).Amuchlargersta-
Arg30. bilization(upto2.32kcal/molat77°C)isachievedbythesingle
Adifferentbehaviorwasobservedforvariant1(Val19/Leu26/ mutationR31L(variant29(Leu31/Glu42)).Finally,thedouble
Ile30).Strikingly,theP26Lsubstitutioninthewild-typeprotein substitution R31L/E42L leads to variant 31 (Leu31/Leu42), a
stronglypromotedtheformationofinclusionbodiesunderall thermophilicversionofHesx-1homeodomain,stabilizedmore
theexpressionconditionstested.Inaddition,theminorfrac- than4kcal/molwithrespecttothewild-typepolypeptide(see
tionofproteinexpressed,whichwassoluble,showedastrong Table1andFig.4a)andcharacterizedbymeltingtemperatures
tendencytoaggregateandprecipitateandwaslostduringthe inthe89–95°Crange.Theoriginofthiseffectcanbeunder-
purificationinallattempts.Therefore,thismutantcouldnotbe stood by examining the thermodynamic data in detail. Thus,
subjectedtoadetailedthermodynamicanalysis.However,tak- despitethedisruptionoftheArg31/Glu42interaction,replace-
AUGUST28,2009•VOLUME284•NUMBER35 JOURNALOFBIOLOGICALCHEMISTRY 23771
SaltVersusHydrophobicBridgesinStabilityandDNABinding
FIGURE4.a,schematicrepresentationofwild-typeHesx-1togetherwithvariants28,29,30,and31withsubstitutionsatpositions31/42.Foreveryvariant,(cid:3)G
valuesat20°C,measuredfromchemicaldenaturationexperiments,arerepresentedinblack.Inaddition,(cid:3)G /(cid:3)G /(cid:3)G termsat77°C,derived
0MNaCl 2MNaCl coulomb
fromthermalunfoldingexperiments,areshowningray.b,thermaldenaturationexperimentsforthewild-typepolypeptideandthethermostablevariant31
monitoredbyone-dimensionalNMR.
ment of both side chains by aliphatic residues results in an stabilityof31(asrevealedbythepersistenceofitsprotonNH
improvement of the homeodomain electrostatic balance (as signals at high temperatures). To determine the influence of
deducedfromthe(cid:3)G term).Inaddition,thesesubstitu- pairLeu31/Leu42onthehomeodomainconformation,itssolu-
coulomb
tions lead to a very favorable (cid:3)G term, suggesting a tion structure was solved by employing NMR methods. An
2 M NaCl
more optimized packing or hydrophobic contacts in the ensemble of 30 structures was calculated on the basis of 703
mutatedproteins.Indeed,energydifferencesat0MNaClareto unambiguousnuclearOverhausereffects.Thestructureshave
a large extent maintained in the presence of very high ionic verysmalldeviationsfromidealgeometryandreasonablenon-
strength(2MNaCl,seeFig.4a).Inprinciple,(cid:3)G should bondedcontacts(supplementalTablesS1andS2).Aschematic
2MNaCl
berathersensitivetothepackingofthealiphaticsidechainat illustrationofthe25simulatedannealedstructuresisdepicted
position 31. To test this point, the leucine residue present in inFig.5andsupplementalFig.S8.
variant31(Leu31/Leu42)wasreplacedbyanisoleucinetogive It can be observed that, despite the mutations, the global
mutant30(Ile31/Leu42).Asexpected,30(Ile31/Leu42)and31 structureof31(Leu31/Leu42)isverysimilartothatreportedfor
(Leu31/Leu42)presentidenticalcoulombiccontributionsto(cid:3)G wild-typeHesx-1(backbonepairwiserootmeansquaredevia-
at 77°C. In contrast, the salt-independent contribution is tion of 0.51 Å between residues 8 and 55). According to the
slightlyreducedinvariant30(Ile31/Leu42).Theobserveddesta- NMRdata,Leu31isburied,presentingclearcontactswithside
bilizationmightreflectpartiallythelowerintrinsichelixpro- chainsofLeu42,Pro26,andPhe49(Fig.5,aandb).Minorstruc-
pensityoftheisoleucinewithrespecttothatofleucine.How- turaldifferencesbetweenthetwopolypeptidesarelocatedon
ever,italsosuggeststhataleucinesidechainestablishesmore theloopbetweenhelicesIandII(Fig.5c).Forexample,inthe
optimizedpackinginteractionswithintheproteinhydrophobic wild-typeprotein,Pro26stacksonthearomaticringofPhe49.As
corethananisoleucine. a result of this interaction Pro26 H(cid:4)protons present unusual
Asanextstep,one-dimensionalNMRspectrawerecollected chemicalshifts(1.04/0.12ppm).Forvariant31(Leu31/Leu42),
forthewild-typeproteinandvariant31(Leu31/Leu42)atdiffer- the Leu31/Phe49 contact induces a slight displacement in the
enttemperatures(Fig.4b).Itcanbeobservedthatthethermo- loop residue Pro26, which is clearly reflected in its side-chain
philicpolypeptideexhibitsgooddispersionintheprotonone- resonances(i.e.theprotonH(cid:4)2isshifteddownfield0.85ppm
dimensional spectra, a characteristic of well folded proteins. withrespecttowild-typeHesx-1).Therefinedstructureofthe
Moreover,thelackofsignificantbroadeningofthesignalsrules thermophilicpolypeptideconclusivelyshowsthattheinterac-
outtheexistenceofaggregationundertheexperimentalcondi- tionbetweenPro26andPhe49(presentinthewild-typehome-
tionsemployed.Finally,theNMRdataareinagreementwith odomain)ispartiallydisruptedin31bytheburialofLeu31side
the CD denaturation experiments confirming the enhanced chain in the hydrophobic core. In contrast to residue 31, the
23772 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER35•AUGUST28,2009
SaltVersusHydrophobicBridgesinStabilityandDNABinding
FIGURE5.a,twodifferentregionsofahomonucleartwo-dimensionalNOESYspectrummeasuredforthethermostablevariant31.Keycontactsbetweenside
chainofLeu31andresiduesPhe49,Pro26,andLeu42arerepresentedinred.b,ensembleof25NMRstructures(right)obtainedforvariant31.Asingle
representativestructureisshownontheleftforclarity.Inallcases,sidechainsofresidues31/42arerepresentedinredandthoseofthecoreresidues20,26,34,
48,and49areshowningreen.c,ensembleof25NMRstructuresofvariant31(backboneisrepresentedingrayandkeysidechainsinyellow)superimposedon
theaveragestructureofwild-typeHesx-1(backboneandsidechainsareshowningreenandred,respectively).ThePro26/Phe49interaction,observedinthe
wild-typehomeodomain,ispartiallydisruptedin31bytheburialofLeu31sidechaininthehydrophobiccore.
leucineatposition42isexposedtothesolvent.However,this thepolypeptideby7.7°Cat0MNaCl(12).Overall,thesestudies
residue contributes significantly to protein stabilization indicatethatasaltbridgeconnecting17and52ismoderatelysta-
through its contact with Leu31. The close proximity between bilizing.Interestingly,closeinspectionofthesequenceinforma-
both side chains is evidenced by several nuclear Overhauser tionavailableforhomeodomainsrevealsaverylowoccurrenceof
effect cross-peaks. In fact, the three-dimensional structure hydrophobicresiduesatsites17or52.Aliphatic(usuallyValor
obtained shows that Leu42 aliphatic side chain significantly Leu) or aromatic (Tyr in all cases) side chains can be found at
reducesthesolvent-accessiblesurfaceofLeu31,whichisalmost position17inlessthan2%ofthesequencesincludedinourdata
totallyburiedinmutant31. set.Althoughaminorfractionofpolypeptidesexhibitsaromatic
In conclusion, aliphatic side chains at positions 31 and 42 residues(Phe,Trp,orTyr)atposition52((cid:9)0.5%)aliphaticamino
provideamoreefficientstabilizationofthehomeodomainfold acids (Val, Leu, or Ile) are totally absent from this site. Finally,
thanthehighlyconservedArg31/Glu42contact.Thisisachieved hydrophobicbridges(involvingeitheraliphaticoraromaticresi-
without promoting significant aggregation of the polypeptide dues) connecting 17/52 were not found in any sequence. This
evenathighconcentrations.TheNMRdataareconsistentwith observation argues against the viability of such interaction in
alocalrepackingofthehydrophobiccoreinthethermophilic homeodomains.Totestthispredictionadditionalmutantswere
protein,whichisinagreementwiththestabilizing(cid:3)G designed.Thus,variants32and33,withpairsLeu17/Tyr52 and
2MNaCl
terms measured for variants 30 (Ile31/Leu42) and 31 Tyr17/Tyr52,respectively,wereproducedandanalyzed.Inspection
(Leu31/Leu42). ofmolecularmodelssuggeststhatbothpairsshouldbesterically
Salt Bridges Versus Hydrophobic Pairs at Positions 17/52— tolerated and could provide stabilizing interactions with the
Thesaltbridge17/52hasbeensubjectedtoseveralstudiesinthe hydrophobiccoreoftheprotein.Theobtainedresultsaresche-
past.Forexample,astabilizationof0.5kcal/molhasbeenreported maticallyrepresentedinFig.6aandTable1.Aminoramountof
fortheK52EvariantoftheEngrailedhomeodomain(inwhicha mutant 33 (Tyr17/Tyr52), insufficient for a detailed thermody-
bridge Lys17/Glu52 can be formed) with respect to the single namicanalysiswaspurifiedafterseveralattempts.However,the
mutantK52A(9).Moreover,ithasbeenshownthatH52Rreplace- measuredthermaldenaturationprofilesareconsistentwithasig-
mentinvnd/NK-2(allowingtheelectrostaticinteractionbetween nificantdecreaseinstability((cid:4)15°Cat0MNaCl).Regardingvar-
sidechainsofGlu17andArg52)increasesthethermalstabilityof iant32(Leu17/Tyr52),thisproteinwasnotexpressedsolubleunder
AUGUST28,2009•VOLUME284•NUMBER35 JOURNALOFBIOLOGICALCHEMISTRY 23773
SaltVersusHydrophobicBridgesinStabilityandDNABinding
destabilizationismaintainedinthe
presenceof2MNaCl((cid:3)(cid:3)G
2MNaCl
(cid:1)1.69kcal/mol).Afractionofthe
observeddifferencein(cid:3)G ,
2MNaCl
estimated in about 0.2 kcal/mol
(32), could be attributed to the
more unfavorable contribution of
the aspartic acid side chain to the
intrinsic helix propensity of the
polypeptide. The remaining 1.49
kcal/moldifferenceissuggestiveof
less optimized packing or hydro-
phobic interactions in 2 (Asp19/
Arg30).Incontrast,(cid:3)G terms
coulomb
are similar in 2 (Asp19/Arg30) and
FIGURE6.a,schematicrepresentationofwild-typeHesx-1,togetherwithvariants32and33withsubstitutions 19 (Glu19/Arg30), which indicates
atpositions17/52.Forvariant32,stabilitycouldnotbeanalyzedduetostrongaggregationandprecipitation that the electrostatic interaction
oftheprotein.MeasuredT values(inoC)forthewild-typeproteinandmutant33areshown.b,schematic
representationofwild-typemHesx-1andvariant19togetherwithvariants2and27,withGlu3Aspsubstitu- between19and30isnotalteredsig-
tionsatpositions19and42,respectively.Foreverysinglemutation,(cid:3)Gvaluesat20°Cmeasuredfromchem- nificantly by the shortening of the
icaldenaturationexperimentsareindicated.Inaddition,(cid:3)G /(cid:3)G /(cid:3)G termsat55°C,derived
0MNaCl 2MNaCl coulomb acidicsidechain.
fromthermalunfoldingexperiments,areshowningray.Freeenergiesaregiveninkcal/mol.
Inasimilarway,thesinglesubsti-
TABLE2 tution E42D in the wild-type polypeptide (to give variant 27)
MeasuredpKavaluesforglutamateandaspartatesidechainsin leadstoamoderatedecreaseinstability(around0.7kcal/molat
mutants12and15
both20and55°C;seeTable1).Again,theobserveddifference
NmMateRdteitrrraotrisonarsewinerdeicpaeterfdo.rmedat5°Cin10mMphosphate,100mMNaCl.Esti- infreeenergybetweenthewild-typeprotein(withpairArg31/
Residue Variant12 Variant15 Glu42)andvariant27(Arg31/Asp42)isratherinsensitivetothe
Glu14 4.41(cid:5)0.10 4.25(cid:5)0.10 ionicstrength,suggestingthatthereducednumberofhydro-
Glu15 3.83(cid:5)0.14 phobicandvanderWallscontactsestablishedbytheshorter
Glu17 3.75(cid:5)0.10 3.95(cid:5)0.11
Glu19 (cid:9)3.9 Asp42sidechainisresponsibleforthiseffect.Inconclusion,our
Asp29 3.52(cid:5)0.08 3.45(cid:5)0.09 resultsindicatethatthepackingofmethylenegroupsfromsalt-
Glu30 4.60(cid:5)0.15
Glu32 5.13(cid:5)0.12 5.16(cid:5)0.13 bridgingsidechainsprovidesignificantfavorablecontributions
Asp33 3.50(cid:5)0.06 3.65(cid:5)0.07 totheglobal(cid:3)G.
Glu37 4.52(cid:5)0.10
Glu41 3.52(cid:5)0.13 3.47(cid:5)0.11 NMR Analysis of pKa Values for Selected Multiple Mu-
Glu42 4.39(cid:5)0.10 4.40(cid:5)0.10 tants—To further dissect the electrostatic contributions to
Asp43 3.77(cid:5)0.08 3.67(cid:5)0.10
homeodomainstability,wemeasuredthepK valuesofacid
Glu62 4.00(cid:5)0.11 4.10(cid:5)0.10 a
Met67 3.40(cid:5)0.06 3.50(cid:5)0.08 residues(GluandAsp)inthefoldedstateforselectedprotein
variants, employing NMR spectroscopy. By monitoring the
anyoftheconditionstested.ItseemsthattheLeu17/Tyr52combi- chemicalshiftdependenceofresonancesadjacenttothetitrat-
nationstronglypromotesproteinaggregation.Altogether,these inggroups,thedegreeofstabilizationofthechargedstatemay
observations suggest that the nonpolar side chains connecting beestimated(33–36).Ouranalysiswasfocusedonquadruple
17/52reducetheproteinstabilityandmightalsoaffectitssolubil- mutants12and15,giventhattheyincludeallthepotentialsalt
ity.Inconclusion,onlypolarresiduesseemtobetoleratedatposi- bridge interactions conserved within this family of proteins
tions17/52. (involving pairs Arg19/Glu30, Glu15/Arg37, Arg31/Glu42, and
StabilizingInfluenceofSideChainPackinginConservedSalt Glu17/Arg52in12andGlu19/Arg30,Arg15/Glu37,Arg31/Glu42,
Bridges—Inadditiontotheirelectrostaticcontributiontothe andGlu17/Arg52in15).Theresultsobtainedarerepresentedin
energyoffolding,chargedsidechainscouldalsocontributeto Table2andsupplementalFig.S9.
protein stability by means of packing interactions involving ThemeasuredpK valuesforGlu17inboth12and15are3.75
a
methylenegroups.Toanalyzethiseffect,twoadditionalvari- and3.95,respectively,whichisconsistentwithamoderatesta-
antswereprepared.Inbothcases,aglutamicacidinvolvedina bilizingeffectofthechargedresidue(inthe0.6–0.8kcal/mol
saltbridgeinteractionwasreplacedbytheshorterasparticacid rangeassumingapK (cid:1)4.4intheunfoldedstate).Incontrast,
a
(seeFig.6b).Thus,theGlu19/Arg30bridgepresentinvariant19 thesidechainofGlu42showsapK closetothosetypicalfor
a
wasreplacedbypairAsp19/Arg30(variant2) Inasimilarway, random coils (4.39 and 4.40 for 12 and 15, respectively) sug-
.
residueGlu42,whichisinvolvedinasaltbridgewithArg31in gesting that, overall, a negative charge at this position has a
thewild-typeprotein,wassubstitutedbyAsp(togive27,see minorcontributiontoproteinstability.
Table1andFig.6b). Accordingtoouranalysis,negativelychargedGlu15inquad-
Accordingtoouranalysis,thesaltbridgeAsp19/Arg30,pres- ruplemutant12isclearlyfavorable(pK (cid:1)3.83).Onthecon-
a
ent in 2 is strongly destabilizing, with respect to pair Glu19/ trary, glutamate Glu30 has a minor destabilizing influence on
,
Arg30 present in variant 19 (see Table 1). This remarkable thepolypeptideconformation(pK (cid:1)4.60).
a
23774 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER35•AUGUST28,2009