NMR Characterization of the Interaction of the Endonuclease Domain of MutL with Divalent Metal ...

13 Pages·2014·1.22 MB·English

NMR Characterization of the Interaction of the
Endonuclease Domain of MutL with Divalent Metal Ions
and ATP
Ryota Mizushima1, Ju Yaen Kim1, Isao Suetake1, Hiroaki Tanaka1, Tomoyo Takai1, Narutoshi Kamiya1,
Yu Takano1, Yuichi Mishima1, Shoji Tajima1, Yuji Goto1, Kenji Fukui2¤, Young-Ho Lee1*
1InstituteforProteinResearch,OsakaUniversity,Suita,Osaka,Japan,2RIKENSPring-8Center,HarimaInstitute,Sayo-cho,Sayo-gun,Hyogo,Japan
Abstract
MutLisamulti-domainproteincomprisinganN-terminalATPasedomain(NTD)andC-terminaldimerizationdomain(CTD),
connected with flexible linker regions, that plays a key role in DNA mismatch repair. To expand understanding of the
regulationmechanismunderlyingMutLendonucleaseactivity,ourNMR-basedstudyinvestigatedinteractionsbetweenthe
CTD of MutL, derived from the hyperthermophilic bacterium Aquifex aeolicus (aqMutL-CTD), and putative binding
molecules. Chemical shift perturbation analysis with the model structure of aqMutL-CTD and circular dichroism results
revealed that tight Zn2+ binding increased thermal stability without changing secondary structures to function at high
temperatures.Peakintensityanalysisexploitingtheparamagneticrelaxationenhancementeffectindicatedthebindingsite
for Mn2+, which shared binding sites for Zn2+. The coexistence of these two metal ions appears to be important for the
function of MutL. Chemical shift perturbation analysis revealed a novel ATP binding site in aqMutL-CTD. A docking
simulation incorporating the chemical shift perturbation data provided a putative scheme for the intermolecular
interactions between aqMutL-CTD and ATP. We proposed a simple and understandable mechanical model for the
regulation of MutL endonuclease activity in MMR based on the relative concentrations of ATP and CTD through ATP
binding-regulated interdomain interactionsbetween CTDand NTD.
Citation:MizushimaR,KimJY,SuetakeI,TanakaH,TakaiT,etal.(2014)NMRCharacterizationoftheInteractionoftheEndonucleaseDomainofMutLwith
DivalentMetalIonsandATP.PLoSONE9(6):e98554.doi:10.1371/journal.pone.0098554
Editor:FreddieSalsbury,Jr,WakeForestUniversity,UnitedStatesofAmerica
ReceivedJanuary1,2014;AcceptedMay5,2014;PublishedJune5,2014
Copyright:(cid:2)2014Mizushimaetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisworkwassupportedbytheJapaneseMinistryofEducation,Culture,Sports,ScienceandTechnology.Thefundershadnoroleinstudydesign,data
collectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
¤Currentaddress:TransGenicInc.KobeResearchInstitute,Chuo-ku,Kobe-shi,Hyogo,Japan
Introduction endonuclease activities of human and yeast MutLa were
demonstrated in the presence of divalent metal ions such as
DNA mismatch repair (MMR) is the process by which post- Mn2+[8,9].
replicative base pair errors are rectified, thereby enhancing the
Since MutL lacks base specificity as an endonuclease, under-
fidelityofDNAreplication100–1000fold[1].MutationsinMMR standingtheregulationmechanismofMutLendonucleaseactivity
genes have been shown to cause hereditary non-polyposis is one of the central themes that has been focused on to expand
colorectalcancer,referredtoasLynchSyndrome[2,3].Although knowledgeonMMR.MutLconsistsofaC-terminaldimerization
significant differences have been reported between Escherichia coli domain(CTD)andN-terminalATPasedomain(NTD)connected
(E. coli) and eukaryotic MMR, the basic concepts of MMR have with disordered linker regions [10,11]. MutL has been shown to
beendevelopedprimarilythroughresearchonE.coli[4,5].MMR form heterodimers and homodimers in eukaryotes and prokary-
startsfromtherecognitionofmismatchedbasepairsbyMutS,and otes,respectively [5].
is followed by the recruitment of MutL by the mismatch:MutS Alterations in the quaternary structure of MutL were assumed
complex. to result from interdomain interactions between the NTD and
Themismatch:MutS:MutLcomplexhasbeenshowntoinitiate CTD/NTD, and the NTD may be involved in regulating
downstream repair machinery in both E. coli and eukaryotes, in endonuclease activity when it binds with metal ions or ATP
which newly synthesized DNA strands are specifically nicked at [11].IsolatedaqMutL-NTDwaspreviouslyshowntostimulatethe
either side of the mismatch [6,7]. However, the mechanisms endonuclease activity of aqMutL-CTD in a zinc-dependent
underlyingdaughterstrandrecognitionandincisionaredecisively manner [12]. A recent study also indicated that MutS coupled
different between E. coli and eukaryotes. While E. coli MMR with the endonuclease activity of MutL stimulated by the DNA
possesses MutH endonuclease, which cleaves daughter strands at mismatch inan ATP-dependent manner, and alsothat theNTD
hemi-methylated GATC sites, eukaryotes and most bacteria lack of MutL primarily mediated the interaction between MutL and
MutH; hence, the counterpart of MutH in eukaryotic and MutS.Therefore,MutSmaystimulatetheendonucleaseactivityof
eubacteria species was a topic for speculation until the latent MutL through interdomain interactions between the CTD and
PLOSONE | www.plosone.org 1 June2014 | Volume 9 | Issue 6 | e98554
ATP-DependentResponseofEndonucleaseActivity
NTD [13]. However, the functional details associated with Sequential Backbone Assignment of aqMutL-CTD
changes in the quaternary structure remain largely unknown aqMutL-CTD samples uniformly labeled with 2H-13C-15N,
[11,12,14,15]. 13C-15N,or15Nwerepreparedusing50mMpotassiumphosphate
Therefore, the intermolecular interactions between MutL and buffer (pH 6.8) containing 100mM KCl, 5mM DTT, 1 mM
variousbindingmoleculessuchasdivalentmetalions,ATP,other EDTA,10%D O,and0.02%NaN .Potassiumphosphatebuffer
2 3
MMRproteins,andDNAbasedonMutLconformationsneedto was used to assign aqMutL-CTD because the dispersion and
be determined in order to examine the regulation of MutL sharpness of the spectra obtained from samples in potassium
endonuclease activity. Recent structural studies using X-ray phosphatebufferwerebetterthanthoseinTris-HClbuffer(Figure
crystallography have provided information on CTD conforma- S1). All NMR measurements for backbone assignments were
tionsinBacillussubtilisMutL(bsMutL).Thestaticcrystalstructures performedat40uCinanAVANCEII-800spectrometerequipped
of the CTD:Zn2+ complex from Neisseria gonorrhoeae and the with a cryogenic probe (Bruker BioSpin, Germany). Data were
complex of CTD from Saccharomyces cerevisiae, with fragments processedbyNMR-Pipe[19]andanalyzedbySparky.Sequential
containing the MIP-box motifs of the Exo1 and Ntg2 proteins backboneassignmentsofaqMutL-CTDwereperformedprimarily
[1,16,17], revealed the detailed interacting sites and binding onuniformly15N-13C-labeledaqMutL-CTD usingasetofthree-
modes of theCTD forpartner molecules. dimensional(3D)heteronuclearcoherencetransfermeasurements
However, the moderate and/or weak intermolecular interac- ofHNCO,HNCACO,CBCACONH,andHNCACB.Histidine-
tions and conformational dynamics of MutL, which are key to specificisotopelabelingwasintroducedbecauseseveralunassigned
understanding MMR in solution, have been difficult to detect by peaks,especiallyaroundH400andH404inthea2-b4loopregion,
X-ray crystallography. Therefore, solution-state NMR is a hampered the complete sequential backbone assignment (Figure
powerful approach used to examine the intermolecular interac- S2).Thismethodallowedustoassignallthreehistidineresidues:
tionsbetweenMutLandothermoleculesbecauseNMRprovides H353, H400, and H404. The remaining unassigned peaks could
residue-based information, even on weak intermolecular interac- be sequentially assigned based on the assignment information of
tionsand proteindynamics insolution. histidine residues and TROSY-HNCACB and TROSY-CBCA-
We here described an NMR investigation of the CTD of CONH measurements of2H-15N-13C-labeledsamples.
aqMutL-CTD,anditsintermolecularinteractionswithmolecules Assignment results were confirmed by the 1H-15N HSQC
suchasZn2+,Mn2+,ATP,andDNA,whichhavebeensuggested spectra of inversely- and arginine/lysine specifically-labeled
to interact with the CTD. Based on the almost complete NMR samples (Figure S3). The final assignment rate was ,97%,
backbone assignment of aqMutL-CTD (,97% of 103 assignable excluding the 7 proline residues and N-terminal residue.
residues),themodelstructureofaqMutL-CTD,moleculardocking Unassigned residues were L317, D335, L397, N398, and R406,
simulations, and NMR titration/relaxation measurements, we whichwerelocatedintheunstructuredloopregionsofthemodel
revealed the intermolecular interaction and binding sites of structureofaqMutL-CTD(Figure1A).Theassignmentresultwas
aqMutL-CTD for binding partners. We further addressed the depicted on the 1H-15N HSQC spectrum of aqMutL-CTD
biological implications of Zn2+ and Mn2+ binding and a possible (Figure 1A) and final resonance assignments were deposited to
mechanical model for the regulation of MutL endonuclease BioMagRes DB(BMRB accessionnumber 11545).
activity.
NMR Relaxation Measurement of aqMutL-CTD and
Materials and Methods Model-free Analysis
15N longitudinal and transverse relaxation times (T and T )
Protein Expression and Purification and 1H-15N steady state NOE values were measured1 using 2a
aqMutL-CTD was expressed and purified as previously
0.4 mM aqMutL-CTD sample in the same buffer used in the
described [12]. E. coli was cultured in minimal media containing
backbone assignment of aqMutL-CTD using a Bruker DRX-500
13C-glucose and 15N-NH Cl as the sole carbon and nitrogen
4 spectrometer equipped with shielded triple-axis gradient triple-
sources,respectively.Thecellbodywascollectedbycentrifugation
resonance probes at 40uC (Figure S4). T was measured with
1
and lysed by sonication in 50mM Tris-HCl (pH 7.8) containing
relaxationdelaysof5,150,300,450,600,800,1000,1200,1400,
500mMNaCl.Aftercentrifugationat18,000rpmfor60min,the 1600, and 1800ms. T was measured with relaxation delays of
2
supernatantwasheatedat70uCfor10min.Theheatedsolution 7.2,21.6,43.2,64.8,86.4,108.0,129.6,151.2,180.0,208.8,and
was again centrifuged and the supernatant was loaded to the 237.0 ms. Single exponential curve fitting was performed using
DEAE column (TOSOH, Tokyo, Japan). The pass solution was Sparky to obtain the values of T and T . 1H-15N NOE values
1 2
then loaded onto the SP-sepharose column (GE-Healthcare were calculated by comparing the peak intensities with and
Biosciences, USA) pre-equilibrated with 50mM Tris-HCl buffer without 1H saturation of 3 s. The global tumbling time (t ) of
m
(pH 7.8) containing 1 mM dithiothreitol (DTT). Ammonium aqMutL-CTDwasestimatedtobe11.7 nsusingtheT /T values.
1 2
sulfate was added to the collected fraction to yield a final By incorporating the three relaxation parameters (T , T , and
1 2
concentration of 1M. The solution was again loaded onto the NOE)andt valuetothemodel-freeanalysiswithTensor2[20],
m
Toyopearl-phenylcolumn(TOSOH,Tokyo,Japan).Thefractions the values of the order parameters (S2) were obtained under the
of aqMutL-CTD were collected and concentrated using an assumption ofisotropic tumblingof theCTD.
Amicon Ultra centrifugal filter (Millipore, Billerica, USA). The
samplesolutionwasthenappliedtothesuperdex75HRcolumn NMR Titrations of aqMutL-CTD with Various Molecules
(GE Healthcare Biosciences, USA) pre-equilibrated with 50mM Changes in the NMR signals of the 1H-15N HSQC spectra of
potassium phosphate buffer (pH 6.8) containing 100mM KCl, uniformly 15N-labled CTD with the addition of other molecules
1 mM DTT, and 1 mM EDTA. The fractions of aqMutL were (Zn2+,Mn2+,ATP,andADP)weremonitoredusingtheAVANCE
collectedandtheirpuritieswerecheckedbySDS-PAGE(.95%).
II-800 or AVANCE III-950 spectrometer (Bruker BioSpin,
Protein concentrations were determined by the molar extinction
Germany) equipped with a cryogenic probe at 40uC. Measure-
coefficients of aqMutL,19005M21cm21 at 278nm [18].
mentswereperformedusing0.1mMCTDin50mMTris-HClor
PLOSONE | www.plosone.org 2 June2014 | Volume 9 | Issue 6 | e98554
ATP-DependentResponseofEndonucleaseActivity
Figure1.Thetwo-dimensionalNMRspectrumandmodelstructureofaqMutL-CTD.(A)Theresultsofthesequentialbackboneassignment
ofaqMutL-CTD(aqMutL )aredisplayedinthe1H-15Nheteronuclearsinglequantumcoherence(HSQC)spectrum.Cross-peaksarelabeledusing
316–425
theone-letteraminoacidcodeandresiduenumber.Thealiasedpeaks(G332andD351)onthe15Naxisareindicatedwith0{0.(B)Themodelstructure
of the homodimer of aqMutL-CTD was generated by Modeller (see the Materials and Methods). The a-helix and b-strand and both termini are
labelled.(C)Thephi(sphere)andpsi(diamond)anglesandorderparameters(S2),ontheleftandrightaxes,respectively,areplottedagainstthe
residuenumber.Thea-helix(arrow)andb-strand(rectangle)predictedusingTALOS+(open)andModeller(closed)areshownabove.(D)Thevalues
ofS2obtainedfromthemodel-freeanalysis(opencircle)andfromTALOS+(solidcircleplusline)areplottedagainsttheresiduenumbers.
doi:10.1371/journal.pone.0098554.g001
PLOSONE | www.plosone.org 3 June2014 | Volume 9 | Issue 6 | e98554
ATP-DependentResponseofEndonucleaseActivity
potassium phosphate buffer (pH 7.0) containing 100mM KCl, identity of the primary sequence (,32%) between aqMutL-CTD
1 mMDTT,0.02%NaN ,and10% D O. andbsMutL-CTD.
3 2
The titration of Zn2+ was performed using the 25mM ZnCl AgraphicalrepresentationoftheputativeMn2+bindingsiteof
2
stock solution. Aliquots of the stock solution were added to the aqMutL-CTD, based on the NMR peak intensity analysis, was
CTDsolutiontoreachtheratiosof[ZnCl ]/[CTD]=0,0.4,0.8, depicted by referencing the previously published X-ray crystal
2
2.0, and 2.4. Similarly, 50mM MnCl of the stock solution was structure of Mn2+-bound protein (PDB ID: 1N8F), which was
2
prepared, and aliquots of the stock solution were added to shown to possess a similar coordination geometry for Mn2+
aqMutL-CTDinphosphatebuffertoreachtheratiosof[MnCl ]/ bindingusingthe3D-motifsearchcapabilityofSwissPdb-Viewer
2
[aqMutL-CTD]=0,0.2,0.4,0.8,2.0,and4.0.Thepeakintensity [23].
ratiowascalculatedusingtheintensityofthepeakswithoutMnCl
2
asareference.Thestocksolutionof100mMATPandADPwas Docking Simulation of the Complex of CTD with ATP
prepared, and aliquots of the stock solution were added to the A docking simulation was performed using myPresto/Sievgene
CTD solution to reach the ratios of [ATP]/[CTD] or [ADP]/ based on the results of the NMR chemical shift perturbation
[CTD]=0,20,40,60,80,and100.TheexcessadditionofATP analysis of ATP binding to aqMutL-CTD [24]. The ligand
over the ratio of 120 resulted in the precipitation of the protein molecule was prepared in Sybyl mol2 file format. The atomic
sample. Synthesized 21-mer singled-stranded DNA, 59- charges of ATP were obtained from the following web site:
GCGGTCATAGTCAAGATACCG-39 (Integrated DNA Tech- [http://www.pharmacy.manchester.ac.uk/bryce/amber/]. The
nologies, Inc.) was annealed to complementary single-stranded force field parameters for the protein were similar to those in
DNA (Integrated DNA Technologies, Inc.) to obtain 21-bp AMBER parm99 [25].
double-stranded DNA (dsDNA) and the stock solution of 6mM
21-bpdsDNAwaspreparedaspreviouslydescribed[21].Aliquots Circular Dichroism (CD) Spectroscopy
of the stock solution of dsDNA were added to 0.1 mM CTD in Far-UVCDspectraof20mM(0.27 mgml21)aqMutL-CTDin
25mM Tris-HCl buffer containing 25mM KCl, 1 mM EDTA,
50mMTris-HCl(pH7.0)including100mMKClwererecorded
1 mM DTT, 0.02% NaN3, and 10% D2O to reach the ratios of in the absence and presence of 40mM Zn2+, 80mM Mn2+, and
[dsDNA]/[CTD]=0, 4,8,and 12.
2 mMATPat40uC,respectively.HeatscanningofaqMutL-CTD
Chemical shift differences in the cross-peaks by titration were
from 40 to 95–100uC was also performed in the absence and
calculated using therelationship:
presence of divalent metal ions and ATP under the same
concentration settings described above by monitoring CD signals
Dd ~hðDd Þ2zð0:158(cid:2)Dd Þ2i0:5 at 220nm at a rate of 1uC min21. CD measurements were
tot HN N performed with a J-720 spectropolarimeter (Jasco, Japan) using a
cell with a light path of 1mm. CD signals between 195 and
where DdHN and DdN are changes in the 1H and 15N chemical 250nmwereexpressedasthemeanresidueellipticity[h](degcm2
shiftsinppm,respectively.Theweightingfactorof0.158wasused dmol21). Temperature was regulated using a PTC-423L Peltier-
to adjust the relative magnitudes of the amide nitrogen chemical
unit (Jasco,Japan).
shift range andtheamide proton chemicalshift range.
Isothermal Titration Calorimetry (ITC) Measurement
Model Structure of aqMutL-CTD and Its Complexes with
CTD solution was dialyzed against 50mM Tris-HCl (pH 7.0)
Zn2+ and Mn2+
containing 100mM KCl and 1 mM b-mercaptoethanol which
The model structure of aqMutL-CTD was built using the usedinsteadofDTTforminimizingbaselinedriftofthermogram.
homologymodelingmethodologybyModeller9.12[22].TheX- ATDandADPweredissolvedbyusingthebufferafterdialysisand
raycrystalstructuresofbsMutL(PDBID:3KDG)anditscomplex their concentrations were determined by UV-vis absorbance. All
with Zn2+ (PDB ID: 3KDK) were used as a template structure, of the samples were degassed for 3 min at 40uC before being
with the secondary structure restraints obtained from the loaded into the calorimeter. Calorimetric experiments were
TALOS+secondarystructurepredictionbasedonNMRchemical performed with a VP-ITCinstrument (GE-Healthcare Bioscienc-
shift assignment values. es, USA) at 40uC. The nucleotide (2 mM ATP or ADP) in the
We further generated the model structures of aqMutL using injection syringe was titrated into 16mM CTD in the ITC cell
bacterial NgoL MutL-CTD homodimer (PDB ID: 3NCV) and (FigureS8).Titrationexperimentsconsistedof39injectionsspaced
eukaryotic PMS1-CTD in the MutLa-CTD heterodimer (PDB- atintervalsof400sec.Theinjectionvolumewas7 mlandthecell
ID:4E4W)foratemplateX-raystructuretoassessthereliabilityof was continuously stirredat 242rpm.
the homology modeling (Figure S10). Then, we compared these
two model structures with the model structure generated using Light Scattering Measurements
bsMutL-CTD. The overall conformation of the model structure
Light scattering of all of the sample solutions was measured at
with the bacterial X-ray structure is quite similar to that with
40uC using a Hitachi fluorescence spectrophotometer F4500
bsMutL-CTD except for a few residues in the a-helix (a2 in the
(Hitachi,Tokyo,Japan)withtheexcitationwavelengthat350nm
model structure based on bsMutL-CTD) and the b-strand (b3 in
(Figure S9). CTD and ATP were dissolved in 50mM Tris-HCl
themodelstructurebasedonbsMutL-CTD).Theslightstructural
(pH7.0)containing100mMKCland1mMb-mercaptoethanol.
differences in the loop structures were also found. On the one
TheamyloidfibrilsofAb peptideswerealsopreparedforthe
hand, the other model structure generated with the eukaryotic 1–40
controls of light scattering of aggregates. Ab peptides were
X-raystructurecriticallydifferswiththelackofthea-helixinthe 1–40
dissolvedandusedfortheformationofamyloidfibrilsasdescribed
C-terminusofCTD(a4inthemodelstructurebasedonbsMutL-
in the previous study [26]. The formation of Ab fibrils was
CTD) and the additional a-helical component in the N-terminus 1–40
confirmedbythefar-UVCDspectroscopyandthioflavinTassay
of CTD. The model structure of aqMutL-CTD generated using
(datanot shown).
bsMutL-CTD was selected for this study due to the highest
PLOSONE | www.plosone.org 4 June2014 | Volume 9 | Issue 6 | e98554
ATP-DependentResponseofEndonucleaseActivity
Results
Characterization of aqMutL-CTD Stereostructures
The 1H-15N HSQC spectrum displayed well-dispersed cross
peaks, which indicated that a homodimer of aqMutL-CTD is a
solid 3D structure at pH 6.8 and 40uC (Figure 1A). The far-UV
CD spectrum of aqMutL-CTD showed a minimum from
,210nmto,220nm,indicatingstructuredsecondarystructures
(Figure2A).AhomologymodelofanaqMutL-CTDconformation
wasproducedusingModeller9.12toobtaindetailedinformation
onthetertiarystructure(Figure1B)[22].bsMutL-CTD(PDBID:
3KDG) [1] was used as the template conformation for modeling
because bsMutL-CTD showed ,32% sequence identity to
aqMutL-CTD, which was sufficient for reliable homology
modeling [27]. The amino acid sequence and conserved motifs
ofvariousMutLfromdifferentspeciesareshown(FigureS5).The
homology model was generated under the constraints of the
secondary structures predicted by TALOS+ [28], estimating
dihedral angles based on the assigned chemical shift information
of C , Ca, C , HN, and 15N of aqMutL-CTD (Figure 1C). The
O b
producedmodelofaqMutL-CTDformedaninvertedhomodimer
with an interface consisting of four b-sheets (b1–b4), each
abundantly incorporating hydrophobic residues, and one a-helix
(a3), domain swapping with each other in the aqMutL-CTD
dimer(Figure1B).Helixa1,thea1–a2loop,andthea2–b4loop
included the conserved motifs DQHA(X) E(X) , ACRISV, and
2 4
CPHGRPI, respectively.
The S2 value ranged from 0 to 1 depending on the degree of
flexibilityandhigherS2valuesreflectedlowerflexibilityinthefast
timescale.Thus,S2couldbeareporterforproteindynamicsand
structure.HighS2values(over0.7)inthea-helixandb-strandand
lowS2values(below0.7)inunstructuredregionsoftheloopsand
terminal parts were consistent with the predicted secondary
structuralelements(Figure1D).Theloopbetweenthea1anda2
helices showed high S2 values of approximately 0.8, which
indicated a rigidbackbone conformation.
Zn2+-Bound aqMutL-CTD Displayed Enhanced
Thermostability
Zn2+ was previously suggested to be involved in the regulation Figure 2. CD measurements of aqMutL-CTD in the presence
ofMutLendonucleaseactivityforproperMMRinvitroandinvivo and absence of binding partners. (A) The far-UV CD spectra of
aqMutL-CTDwithout(black)andwithZn2+(red),Mn2+(purple),andATP
[1,8,9]andisrelatedtotheinterdomaininteractionsbetweenthe
(green) are shown. Noisy data points in the shorter wavelength from
CTD andNTD [12,15]. 220nminthepresenceofATPwereomitted.(B)HeatscansofaqMutL-
The NMR-based titration of Zn2+ was performed to examine CTD are shown without (black) and with Zn2+ (red), Mn2+ (blue), and
the intermolecular interactions between Zn2+ and aqMutL-CTD ATP(green)usingtheCDsignalat220nm.
insolution.Weobservedlargechangesinthepeaksofthe1H-15N doi:10.1371/journal.pone.0098554.g002
HSQCspectrum:manynewpeaksappearedprimarilybecauseof
slowexchangesandsimultaneouslybroadenedduetointermediate
structures (Figure 2A). The thermal denaturation of CTD in the
exchanges; however, we could not assign all new peaks. presenceandabsenceofZn2+wasexaminedbytracingCDsignals
Representative residues in the slow exchange regime, C402 and
at 220nm (Figure 2B). The results of thermal denaturation
G405, are indicated by rectangles (Figures 3A and 3B). These experiments demonstrated that Zn2+ binding significantly en-
results suggested that Zn2+ binding was tight and related to the
hancedthethermostabilityofaqMutL-CTD.FreeCTDbeganto
relativelybroadregionsofaqMutL-CTD,whichmaybeinvolved
melt cooperatively at ,50uC and was almost completely
in a conformational change. The putative Zn2+ binding sites of denatured at ,95uC. Meanwhile, Zn2+-bound CTD largely
aqMutL-CTDindicatedbythereportedX-raycrystallographyof
changedthethermaldenaturationprofile.CTDmeltedgradually
bsMutL-CTD were also illustrated (Figure 3C). The modeled
from ,65uC, and heat denaturation was not finished even at
complex structure of the CTD and Zn2+ provided putative
,95uC.
residuesthatinteractedwiththetwoZn2+ions:H353andE357in
DQHA(X) E(X) of the a1 helix, C371 in ACRISV, and C402,
2 4 Determination of the Mn2+ Binding Sites of aqMutL-CTD
H404, andR406intheCPHGRPI motif.
in Solution
Thefar-UVCDspectrumofaqMutL-CTDwasobtainedinthe
presence of Zn2+ to further characterize the Zn2+-bound state of Recent endonuclease assays have suggested that Mn2+ is
aqMutL-CTD. No change was observed in the CD spectrum, essential to activate the endonuclease activity of aqMutL-CTD
which revealed that Zn2+ binding did not perturb the secondary
PLOSONE | www.plosone.org 5 June2014 | Volume 9 | Issue 6 | e98554
ATP-DependentResponseofEndonucleaseActivity
Figure3.ThecharacterizationofinteractionsbetweenaqMutL-CTDandZn2+.(A)The1H-15NHSQCspectraof100mMaqMutL-CTDatthe
variousconcentrationsofZn2+aresuperimposed.Therepresentativepeaks(C402andG405)showingtheslowexchangeareindicatedwiththeone-
letteraminoacidcodeandresiduenumber.(B)ThechangeinthechemicalshiftofC402andG405.(C)PutativebindingsitesofZn2+aremagnified
(seetheMaterialsandMethods).ThesidechainsofH353,E357,C371,C402,andH404areshown.ThebluespheresindicateZn2+ions.
doi:10.1371/journal.pone.0098554.g003
invitro[12].Otherstudieshavealsoclarifiedthathumanandyeast together with information on the characteristic coordination
MutLashareadependence onMn2+endonucleaseactivity[8,9]. geometry of Mn2+ in the Mn2+-bound protein [30], we coordi-
NMR-based analysis was performed to examine the Mn2+ nated Mn2+ around E357, C402, and H404 to visualize Mn2+
binding site of aqMutL-CTD at the residue level in solution. On binding sites (Figure4D).
thebasisoftheeffectoftheparamagneticrelaxationenhancement Far-UV CD measurements were performed to investigate the
(PRE) of Mn2+, we expected to detect even transient and weak effectsofMn2+bindingonchangesinthesecondarystructureand
intermolecular interactions by observing a decrease in the peak thermal stability (Figure 2). Although the far-UV CD spectrum
intensitybecausePREwaspreviouslyshowntobeproportionalto was almost identical to that in the absence of metal ions
the inverse sixth power of distance [29]. Although no significant (Figure 2A), the thermal transition curve was slightly different
perturbationwasdetectedinthechemicalorpseudo-contactshift (Figure 2B). Mn2+ binding shifted the transition curve to slightly
(Figure4A),markeddecreaseswereobservedintheNMRsignals higher temperatures. These results implied that Mn2+ interacted
ofCTDwiththeadditionofMn2+(Figure4B).Overalldecreases less specifically and strongly with CTD than with Zn2+, which
inthepeakintensityimpliedthatthewholemoleculeofaqMutL- were consistent withtheNMRresults.
CTD was susceptible totheeffects ofthesolventPRE of Mn2+.
Large decreases in intensities were observed in several InvestigationofIntermolecularInteractionsbetweenATP
characteristic regions (peak intensity ratio ,0.52): the region of and aqMutL-CTD
a1includingtheDQHA(X) E(X) motif,thea1–a2loop,andthe AlthoughATPbindingtoMutLiskeytoMMR,intermolecular
2 4
a2-b4 loop including the CPHGRPI motif (Figures 4B and 4C). interactionsbetweentheCTDandATPremainlargelyunknown
Among them, a marked decrease in the peak intensity ratio (, [11,12,14,15,31,32]. Therefore, we obtained residue-based infor-
0.22) was observed in L354, E357, C402, and G405. Taken mationonATPbindingusingNMRspectroscopy.Thetitrationof
PLOSONE | www.plosone.org 6 June2014 | Volume 9 | Issue 6 | e98554
ATP-DependentResponseofEndonucleaseActivity
Figure4.Characterizationof interactionsbetweenaqMutL-CTDandMn2+.(A)The1H-15NHSQCspectraof100mMCTDatthevarious
concentrationsofMn2+aresuperimposed.(B)ThepeakintensityratioofCTDintheabsenceandpresenceof400mMMn2+wasplottedagainstthe
residuenumbers.Theuppersolidandmiddledashedlinesrepresent0meanvalue(0.52)0and0meanvalue2standarddeviation(0.37)0.Thebottom
dottedlinesignifies0meanvalue226standarddeviation(0.22)0.(C)Mn2+bindingsitesbasedonthepeakintensityratioinBareshownwiththe
colorcode:red,peakintensityratio,0.22;yellow,0.22,peakintensityratio,0.37.(D)ThebindingsitesforMn2+aremagnified.Thepurplesphere
indicatesMn2+.
doi:10.1371/journal.pone.0098554.g004
ATP against the CTD showed the shift of peaks with a fast chemical shift can be perturbed by the changes in electronic
exchange regime, which revealed the intermolecular interactions environments around NMR-active nuclei of interest. Therefore,
between the two molecules with weak affinity (Figure 5A). NMR severalfactorsinthebindingsysteminduceperturbationsofNMR
PLOSONE | www.plosone.org 7 June2014 | Volume 9 | Issue 6 | e98554
ATP-DependentResponseofEndonucleaseActivity
chemical shifts such as the ligand binding without the conforma- ATP shares a binding site with DNA, endonuclease activity of
tionalchange and/ortheligand bindingwiththeconformational CTD should beaffected by thepresenceof ATP.
change. It is often difficult todistinguish the mixed factors which However,therecentstudybyShimadaetal.demonstratedthat
are attributed to the apparent change in the chemical shift using Mn2+-dependent endonuclease activity of Thermas thermophilus
onlyNMRdata.Therefore, althoughwecannotasserttheorigin MutL (ttMutL)-CTD did not depend on the presence of ATP.
ofalotofchemicalshiftchangesofaqMutL-CTD,ATPobviously Endonuclease activity of ttMutL-CTD in the absence and
binds to aqMutL-CTD and ATP-binding may induce structural presence showed same activity [13]. Moreover, Mn2+-dependent
rearrangements. endonucleaseactivityofaqMutL-CTDwasalsonotstimulatedor
Thedetailedanalysisfortheaffinitybasedonthechemicalshift inhibitedbyAMPPNP(FukuiK.,unpublisheddata).Theseresults
perturbation was hampered by the protein aggregation in the indicate that ATP does not competitively bind to the dsDNA
presenceofthehighATP-concentrationover12mM.Itshouldbe binding siteof MutL-CTD withDNA,andvice versa.
noted that examining of CTD aggregation is important to Taken alltogether, thebinding siteonaqMutL-CTD forATP
understand endonuclease activity of MutL-CTD. Therefore, we which was revealed in this study is novel and is not used for the
compared the intensity of CTD at each ATP concentration to DNA binding(i.e., an active siteof thenuclease).
examine the formation of aggregates. The results showed the
comparable intensities: the NMR peak intensity of CTD in the Investigation of ATP Binding to aqMutL-CTD
presence of ATP with the 100-fold higher concentration than
In order to obtain more information on binding specificity of
CTD did not decrease compared to the peak intensity of CTD
ATPtoaqMutL-CTD,wefurthercarriedouttheNMRtitration
without ATP, indicative of the absence of aggregation. This is
experimentsonADPbindingtoaqMutL-CTD(FigureS7)andthe
because, in general, the formation of aggregates decreases
ITC measurements on both ATP and ADP binding to CTD,
significantly the intensity of the NMR signals. In order to obtain
respectively (Figure S8).
more evidence, we additionally performed the light scattering
A series of the NMR titration spectra of aqMutL-CTD with
measurements of CTD in the absence and presence of ATP
ADP revealed the large differences from those with ATP.
(FigureS9).Highandlowintensityoflightscatteringindicatelarge
Although the ADP titration spectra also showed the changes of
and insoluble aggregates as well as small and soluble proteins,
NMRpeaks,thenumberofperturbedpeaksforADPtitrationwas
respectively. For the reference intensity of aggregates, we also
lessthanthatforATPtitration.Interestingly,allofthetrajectories
carried outthe light scattering measurement of the amyloid fibril
of the shifted peaks upon ADP titration including the peaks (e.g.
of Ab peptides which is an ordered aggregate. As shown in
1–40 G405,Y409,andG422)whichshowedthetwodifferentdirections
Figure S9,theintensitiesof CTD intheabsenceand presenceof
of the trajectory of the peak shift upon ATP titration were linear
ATPweremuchlowerthanthoseoftheamyloidfibrilsofAb
1–40 (Figure S7A). In addition, the magnitude of chemical shift
peptides and were similar to the intensity of monomeric Ab
1–40 perturbation on ADP titration was lower than that on ATP
peptides.Allofthesedataindicatedtheabsenceofaggregationof
titration (Figure S7B). These results indicate that ADP also binds
CTD in the presence of excess amounts of ATP but less than
to aqMutL-CTD (Figure S7C) with the lower specificity and
12mM.
binding affinity than that forATPbinding.
Chemical shift perturbation analysis also revealed the binding
At the same time, we can also exclude the possibility that
sitesforATP.Theresiduesina1(K365),theloopbetweena1and
perturbationofpeaksbyATPandADPtitrationwasnotinduced
a2 (I373 and S374), and the C-terminal parts (G405, I408, and
bytheionicstrengthchangeofNMRsolventbyadditionofATP
V421)showedahighchemicalshiftperturbation(CSP)over0.082
and ADP. If there is an influence from the ionic strength of the
onATPbinding(Figure5B).Theperturbedresiduesweremapped
on the model structure depending on the CSP values using the addednucleotides,theshiftofNMRpeaksofaqMutL-CTDupon
color codes (Figure 5C). additionofATPorADPshouldshowasimilarpattern.However,
as described above, the pattern of trajectory between ATP and
The docking simulation, which incorporated the CSP data,
provided a putative scheme for the intermolecular interactions ADPtitrationwasobviouslydistinct,indicativeofspecificbinding
between the CTD and ATP. The results obtained indicated that of ATPandADPtoaqMutL-CTD.
ATP was bound to the cleft formed by a1 including the ITCresultsgaveafurtherinsightintoATPandADPbinding.
DQHA(X) E(X) E motif, the a1–a2 loop including the ACRISV The calorimetric titration showed endothermic reaction heat for
2 4
motif, and the a2–b4 loop including the CPHGRPI motif titrating ATP or ADP to aqMutL-CTD (Figure S8). The
(Figure5D).ThesidechainsofR358,K365,andN398interacted magnitude of observed heat for ATP titration was greater than
electrostatically with the b-, a-, and c-phosphates of ATP. The thatforADPtitrationbyapproximatelyfive-fold,indicatingmore
riboseandadenineofATPwereboundprimarilybeneaththea2– tightintermolecularinteractionsbetweenATPandaqMutL-CTD
b4loop. than those between ADP and CTD. Both titration was not
Thefar-UVCDspectrumandthermaltransitioncurveofCTD saturatedatthemolarratio([nucleotide]/[aqMutL-CTD])ofeven
in the presence of ATP were identical to those without ATP, 25,consistentwiththeresultsofNMRtitration.Mostimportantly,
indicating that weak ATP binding did not change the secondary theendothermic peaks forbothtitration decreased graduallyand
structure andglobal thermal stability(Figure 2). thepeaksofATPtitrationweremorerapidlydecreasedthanthose
Finally, to rule out the possibility of sharing binding sites for forADPtitration.Althoughthebindingcurvesweretoogentleto
DNA, i.e., competitive binding between DNA and ATP, we analyze the data precisely, these ITC data suggested that ATP
directly measured the binding of DNA to aqMutL-CTD. As binding is more specific than ADP binding with high binding
showninFigureS6,excessamountsofDNArelativetoaqMutL- affinity compared to ADP binding. The different binding affinity
CTD did not give any changes in the NMR signals of aqMutL- andspecificityofATPandADPtoMutL-CTDmaycomefroma
CTD,indicatingthatbindingaffinityofDNAtoaqMutL-CTDis phosphate.
extremely low. Although a binding region for DNA was not ItshouldbenotedthatfurtherbindingassaysusingGTP,NTP,
determined experimentally, it is obvious that binding affinity of ordATParerequiredtoobtainaninsightintomolecularoriginon
aqMutL-CTDforATPishigherthanthatforDNA.Asaresult,if binding specificity ofATPtoMutL-CTD.
PLOSONE | www.plosone.org 8 June2014 | Volume 9 | Issue 6 | e98554
ATP-DependentResponseofEndonucleaseActivity
Figure 5. The characterization of interactions betweenaqMutL-CTDand ATP. (A) The 1H-15N HSQC spectra of 100mM CTD at various
concentrationsofATParesuperimposed.Theresiduesdisplayingsignificantperturbationsarelabeledusingaone-letteraminoacidcodeandresidue
numbers.PeaksofC402(?)disappearedafter[ATP]/[CTD]=80asthetitrationproceeded;itwasthenruledoutinthechemicalshiftperturbation
(CSP)analysis.However,thelabelwasdisplayedforthediscussionbelow.(B)CSPsoftheCTDinthepresenceof8mMATPwereplottedagainst
residuenumbers.Thebottomsolidlinerepresentsthe0meanvalue(0.045)0.Themiddleandupperdottedlinessignify0meanvalue+0.56standard
deviation(0.064)0and0meanvalue+standarddeviation(0.082)0forsignificantchanges,respectively.(C)Thedegreeoftheperturbationwasmapped
PLOSONE | www.plosone.org 9 June2014 | Volume 9 | Issue 6 | e98554
ATP-DependentResponseofEndonucleaseActivity
ontothemodelstructurewiththecolorcode:Red,CSP.0.082,yellow,0.082.CSP.0.064.(D)ThecomplexstructureoftheCTDandATPasobtained
bythedockingsimulationusingMyPresto/Sievgene(seetheMaterialsandMethods).Theinteractingsitesareenlarged(right).ATPandaqMutL-CTD
arerepresentedbyaballandstickmodelandmeshedsurfacemodel,respectively.
doi:10.1371/journal.pone.0098554.g005
Discussion (E357K and H404A) displayed the inactivation of the Mn2+-
dependentincisionofsupercoiledDNA,whichmayhavebeendue
We here demonstrated that Zn2+, Mn2+, and ATP bound to todecreasesinthebindingcapabilityoftheCTDforMn2+[41].
aqMutL-CTD without alternating the secondary structure. Only These findings support the results of the present study in which
Zn2+ markedly increased thermal stability, and this has been E357 and H404 consisted of binding sites for Mn2+, as shown in
attributed to changes in tertiary structures and/or dynamics. It Figure 4B.
should be noted that although the complete profile of thermal Mn2+ shared CTD binding sites with Zn2+, with similar
unfolding of aqMutL-CTD in the presence of Zn2+ was not coordinationgeometries(Figures3and4).BecauseZn2+enhanced
obtained, it is obvious that cooperativity of thermal unfolding of theendonucleaseactivityofbsMutLinthepresenceofMn2+[1],
Zn2+-bound aqMutL-CTD is higher than that of apo-CTD, these two divalent metal ions may be coordinated together for
indicating the increase in thermal stability. Many new NMR MMR invivo to ensure both stability and function; however,
signals can be interpreted by reorganizing large parts of the detailed future work is required to clarify these intermolecular
tertiary structure (Figure 3A). Several key residues suchas H353, interactions.
E357, C371, C402, and H404 may be important for suppressing ATP binding to MutL and MutLa for MMR has been
global motion due to the tight binding of Zn2+ (Figure 3B). demonstrated and the NTD was predominantly suggested to
PreviousstudiesalsoreportedthatZn2+ionsservedasastructural accommodate ATP [14,31]. However, there has been little focus
factor rather than a catalytic contribution [33–35], implying a on ATP binding to the CTD and no consensus on their
relationship between Zn2+ binding and stability. Since aqMutL- intermolecular interactions. Mauris J. et al. showed that ATP did
CTD functions at high temperatures above 90uC, Zn2+ binding not bind aqMutL-CTD using the filter binding assay and further
shouldbeessentialformaintainingconformationalstabilityforits indicatedtheabsenceofATPbindingmotifinaqMutL-CTD[41].
function. Incontrast,itcouldbeimpliedthattheintermolecularinteractions
Mn2+hasbeenshowntoplayapivotalroleintheendonuclease betweenCTDfromttMutLandATPmaybepossible[42].This
activityofaqMutL-CTD.Althoughithasbeensuggestedthatthe discrepancy may reflect the difficulty in detecting weak intermo-
coordinationnumberandresiduecompositionoftheZn2+-binding lecular interactions, which are markedly susceptible to subtle
site of MutL may preclude Mn2+ binding based on the changes in the surrounding conditions used such as temperature,
bioinformatics of metal-binding proteins [35–40], we clarified salt concentration, andpH.
thattheMn2+bindingsitesofaqMutL-CTDwerelocatedaround We here demonstrated weak and specific interactions between
the endonuclease activity sites between the two motifs, ATP andaqMutL-CTD using solution-state NMR at the residue
DQHA(X) E(X) E and CPHGRPI (Figure 4). The reported level and ITC at the molecular level. By incrementally adding
2 4
endonuclease activity assay of the aqMutL-CTD mutants excess ATP to aqMutL-CTD, we revealed gradual shifts in peak
positions, which indicated weak intermolecular interactions. The
linearshiftfollowedsuccessivelybyalinearshiftinadifferentway,
as represented by G405, I408, and Y409 (Figure 5), implied the
two states of the ATP-bound CTD. Considering the concentra-
tionsofATPandCTDusedhere,theaffinityoftheCTDforATP
was markedly lower than that of the NTD, the dissociation
constantofwhichisapproximately20nM(FukuiK.,unpublished
data). ITC data also showed the weak affinity of CTD for ATP,
consistent withtheNMRresults.
Chemicalshiftperturbationanalysisandthedockingsimulation
allowedustodeterminethebindingsitesforATP,whichincluded
themotifsconservedinthevariousspecies(FigureS5),andimplied
that the binding of ATP to the CTD may be a common feature.
The feature of the rigid conformations of the a2–b4 loop
containingtheconservedCPHGRPImotifduetohydrogenbond
networks (Figure 1D) may be involved in the effective binding of
Figure6.Activation-attenuationmodelfortheATPconcentra- metal ions and ATP. Interestingly, the ATP binding sites of
tion-dependent response of MutL endonuclease activity. The aqMutL-CTD were included in the regions responsible for the
threeconformationalstatesofMutL(rest,active,andinhibitedstates) interdomaininteractionsfortheNTD,whichhavebeensuggested
are in equilibrium. The dominantconformational state can be shifted
topromotetheendonucleaseactivityoftheCTD[12,13,15].This
depending on the differences in the relative concentration between
indicatesthepossibilityofregulatoryroleofATPbindingtoCTD
MutL and ATP. The rest state of MutL in the absence of ATP (left) is
predominantlypopulated.ThesmallincreaseintheATPconcentration inCTD-NTD interdomain interaction.
drives the ATP binding to NTD thereby stabilizing the active state In addition, Fukui K. and coworkers modified Cys496 of
(middle).AtthehighATPconcentration, however,theinhibitedstate ttMutL-CTD, which is the conserved cysteinyl residue in
whichsuppressesendonucleaseactivityisfavoredbyaccommodating
CPHGRPI motif corresponding to Cys402 of aqMutL-CTD
further ATP to CTD (right). It should be noted that the complicated
[42]. They showed that chemical modification of Cys496 with
conformationalstatesofATP-boundCTDwhichareATPconcentration-
dependent are not considered for simplicity. The ellipses and circles DTNB and the substitution of Cys496 with an Ala residue in
indicatetheCTDandNTD,respectively.BluerectanglesshowATP. ttMutL caused the decrease in the efficiency of AMPPNP-
doi:10.1371/journal.pone.0098554.g006 dependent suppression of endonuclease activity. Consideringthat
PLOSONE | www.plosone.org 10 June2014 | Volume 9 | Issue 6 | e98554