Molecular Dynamics Simulations Show That Bound Mg Contributes to Amino Acid and Aminoacyl ...

13 Pages·2006·0.47 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.281,NO.33,pp.23792–23803,August18,2006
©2006byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
Molecular Dynamics Simulations Show That Bound Mg2(cid:1)
Contributes to Amino Acid and Aminoacyl Adenylate Binding
Specificity in Aspartyl-tRNA Synthetase through Long Range
Electrostatic Interactions*□S
Receivedforpublication,March27,2006,andinrevisedform,June12,2006 Published,JBCPapersinPress,June14,2006,DOI10.1074/jbc.M602870200
DamienThompson‡§1andThomasSimonson‡2
Fromthe‡LaboratoiredeBiochimie,CNRS,UMR7654,DepartmentofBiology,EcolePolytechnique,91128Palaiseau,
Franceand§TyndallNationalInstitute,UniversityCollegeCork,Cork,Ireland
Molecularrecognitionbetweentheaminoacyl-tRNAsynthe- important class of information-processing enzymes (1–4).
taseenzymesandtheircognateaminoacidligandsisessential EachaaRScatalyzestheaminoacylationofaspecifictRNAbya
forthefaithfultranslationofthegeneticcode.Inaspartyl-tRNA cognate amino acid, establishing the genetic code (5–7). The
synthetase(AspRS),theco-substrateATPbindspreferentially amino acid (aa) and ATP react first to form an aminoacyl
withthreeassociatedMg2(cid:1)cationsinanunusual,bentgeome- adenylate;inasecondstep,theaminoacidistransferredtothe
try.TheMg2(cid:1)cationsplayastructuralroleandarethoughtto tRNA. Some aaRSs have evolved a third, editing step where
alsoparticipatecatalyticallyintheenzymereaction.Co-binding incorrecttRNA-aaproductsarehydrolyzed(8–10).Specificity
oftheATP(cid:2)Mg32(cid:1)complexwasshownrecentlytoincreasethe for the aa and the tRNA can arise from different component
Asp/Asnbindingfreeenergydifference,indicatingthatamino steps,suchasbindingorreleaseoftheaminoacidorbindingor
aciddiscriminationissubstrate-assisted.Here,weusedmolec- acylation of the tRNA. Furthermore, through their particular
ulardynamicsfreeenergysimulationsandcontinuumelectro- combination of reversible binding and irreversible reaction
static calculations to resolve two related questions. First, we
steps,aaRSscanusespecificityinsuccessivestepstoamplifythe
showedthatifoneoftheMg2(cid:1)cationsisremoved,theAsp/Asn
overalleffect(11).Forexample,aminoacidspecificitycanbe
binding specificity is strongly reduced. Second, we computed establishedintheaaRS(cid:2)aacomplex,theaaRS(cid:2)aaAMPcomplex,
therelativestabilitiesofthethree-cationcomplexandthe2-cat-
orboth.
ion complexes. We found that the 3-cation complex is over-
The20aaRSsformtwodistinctclassesof10memberseach
whelminglyfavoredatordinarymagnesiumconcentrations,so
(6).Belowwehavefocusedmainlyonaspartyl-tRNAsynthetase
thattheproteinisprotectedagainstthe2-cationstate.Inthe
(AspRS),oneofthebeststudiedaaRSs.AspRSbelongstothe
homologous LysRS, the 3-cation complex was also strongly
aaRS class II, forming a subclass IIb with AsnRS and LysRS.
favored,butthethirdcationdidnotaffectLysbinding.IntRNA-
Although aaRSs are generally very amino acid-specific, they
bound AspRS, the single remaining Mg2(cid:1) cation strongly
haveacomplexevolutionaryhistory(4,12),whichhasledtoa
favoredtheAsp-adenylatesubstraterelativetoAsn-adenylate.
remarkablediversityinthemodernenzymes.WithinclassIIb,
Thus, in addition to their structural and catalytic roles, the
forexample,LysRSisveryspecificinyeast;butinEscherichia
Mg2(cid:1) cations contribute to specificity in AspRS through long
coli,itismorepromiscuous(13).InE.coli,AspRSdiscriminates
rangeelectrostaticinteractionswiththeAspsidechaininboth
stronglyagainstAsn,butmoreweaklyagainstD-Asp(10).Sev-
thepre-andpost-adenylationstates.
eralaaRSsachieveahighfidelitythroughtheireditingstep.An
ambiguousIleRSwasconstructedrecentlybydeletingtheIleRS
editingdomain(14);theresultingIle/Valambiguityactuallyled
Specificmolecularassociationisfundamentaltomanybio-
to a growth advantage in bacteria. As a last example, many
chemical processes and is frequently used to transfer energy
archaebacteria lack AsnRS and produce tRNAAsn-Asn by an
or information. Aminoacyl-tRNA synthetases (aaRSs)3 are an
indirect route; tRNAAsn is aspartylated by a “nondiscriminat-
ing” AspRS (which accepts both tRNAAsp and tRNAAsn), and
*Thecostsofpublicationofthisarticleweredefrayedinpartbythepayment
thentheAspmoietyisamidated(1).
ofpagecharges.Thisarticlemustthereforebeherebymarked“advertise-
ment”inaccordancewith18U.S.C.Section1734solelytoindicatethisfact. Themechanismofthetwoenzymereactions,aminoacid
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains adenylation and tRNA aminoacylation, is qualitatively
supplementalmaterialincludingFig.SM1andTableSM1.
1SupportedinpartbyanEgidepostdoctoralfellowship. understood in both aaRS classes (1). For AspRS, crystal
2Towhomcorrespondenceshouldbeaddressed:LaboratoiredeBiochimie structuresareavailablefromseveralorganisms,encompass-
(CNRS,UMR7654),Dept.ofBiology,EcolePolytechnique,91128Palaiseau, ingthethree“kingdoms”oflifeandthewholereactionpath-
France. Tel.: [phone]; Fax: [phone]; E-mail: thomas.
way: apoenzyme, complexes with Asp alone, ATP alone,
[email protected].
3The abbreviations used are: aaRS, aminoacyl-tRNA synthetase; aa, amino aspartyl-adenylatealone(AspAMP)(15–20),andcomplexes
acid(s);AspRS,aspartyl-tRNAsynthetase;AMP,adenosinemonophosphate; withtRNAAsppresent(21–23).Eachsubstrateisrecognized
ATP, adenosine triphosphate; MD, molecular dynamics; MDFE, molecular
by side chains conserved throughout AspRSs (Asp side-
dynamicsfreeenergy;PBFE,Poisson-Boltzmannfreeenergy;PME,particle
meshEwald;CRF,continuumreactionfield;r.m.s.,rootmeansquare. chainrecognition)orthroughoutallormostofclassII(ATP,
23792 JOURNALOFBIOLOGICALCHEMISTRY VOLUME281•NUMBER33•AUGUST18,2006
This is an Open Access article under the CC BY license.
CationBindingandAspRSSpecificity
Asp backbone recognition). The active site is highly preor- latedstatesaredifficulttostudyexperimentally.Oneextensive
ganized to receive the Asp and ATP substrates, with the mutationstudyexploredchangesintheapparentAspandATP
apoenzyme largely superimposable on the various complex dissociation constants spanning only 2 orders of magnitude,
structures (22). Each substrate, taken separately, is almost correspondingtoafreeenergyspanoflessthan3kcal/mol(26).
exactlysuperimposableonthecorrespondingmoietyinthe Non-cognatecomplexeslikeAspRS(cid:2)Asncouldnotbeanalyzed.
known AspAMP or tRNA(cid:2)Asp complexes. In all of class II, Weakly populated states are invisible in a crystal structure.
ATPbindsinaveryunusual,completelybentconformation, Thus,fromtheAspRS(cid:2)ATPcrystalstructures,ATPbindspref-
withthreeassociatedMg2(cid:1)cations(ortwoinafewcases;see erentially with three associated Mg2(cid:1) cations (17). However,
below). The principal cation coordinates both the reactive thecrystallographicdatadonotrevealtheexactoccupanciesof
(cid:1)-phosphateandthe(cid:2)-phosphateandispositionedbycon- thethreemagnesiumsites.AcomplexwithATPandonlytwo
servedsidechains(24).Theothertwocationscoordinatethe cations,presentinthecrystalwithapopulationof10or20%,for
(cid:2)-and(cid:3)-phosphatesoneithersideoftheATP. example,wouldnothaveanappreciableeffectontheelectron
InAspRS,theadenylationreactioncanoccurintheabsence densitymapsandwouldbeimpossibletoinferfromthecrys-
of tRNA. Once Asp and ATP are in place, the Asp backbone tallographicdata.
reactswiththe(cid:1)-phosphatethroughanin-linemechanismand Theoreticalmethodsrepresentavaluablecomplementary
a pentacoordinate transition state. Inversion of the (cid:1)-phos- toolthatcanresolvethesedifficulties(32–37).Thespecific-
phate is clearly seen when crystals of AspRS(cid:2)ATP and ityofAspbindingtoAspRSisgovernedbythebindingfree
AspRS(cid:2)AspAMParecompared(19).Aswithmostenzymereac- energy difference between the cognate Asp and competitor
tions,theexactroleofeachsurroundinggroupishardtoestab- ligands. This difference can be obtained from molecular
lish.ItispresumedthattheprincipalMg2(cid:1)cationhelpsacti- dynamics free energy simulations (MDFE), which have
vatethe(cid:1)-phosphatebywithdrawingelectronsandpullingits matured enormously in recent years and have been used to
oxygensintoapentacoordinategeometry,helpingtostabilize study several aaRSs. Extensive studies (33–35) show that
the transition state through electrostatic interactions. The whentheAspRSbindingpocketisinthe“open”state(open
othertwocationsneutralizetheleavingpyrophosphateproduct flippingloop),thereisanenormouspreferenceforAspover
andmayalsocontributetotransitionstatestabilization. Asn,thankstoanetworkofelectrostaticinteractionsinthe
WehavefocusedhereontheroleofMg2(cid:1)inAsp/Asndis- active site. A thermodynamic cycle (see below) was used to
criminationbyAspRSfromE.coli.AspRSspecificityisacom- obtainbindingfreeenergydifferences,andagroupdecom-
plexproblem.Althoughtheenzymeispreorganized,Aspbind- positionofthefreeenergywasusedtoidentitytheresidues
ing does induce structural reorganization in two important determiningaminoacidbindingspecificity.
regions: (i) a so-called histidine loop (residues 436–449 in Whentheflippingloopcloses(17,23),thenegativeGlu-171
E.coli)shiftsandbecomesmoreordered,withHis-448making isbroughtclosetotheAspligandsite.Werecentlypredicted
ahydrogenbondtoAsp;(ii)aflexible“flippingloop”(residues computationally(36)thatthisconformationalchangeinduces
167–173inE.coli)closesovertheaabindingsite,bringingthe proton binding by the nearby His-448. The His-448 positive
negativeGlu-171closetotheAspligand.Theflippingloopis chargethenaccountsformostofthelarge,computedAsp/Asn
conservedineukaryal,eubacterial,andarchaebacterialAspRS discrimination.Inanotherlongrangeelectrostaticeffect,asub-
(17,18,23),andacorrespondingmobileloopisfoundinAsnRS strate-assisted specificity was observed: co-binding of ATP
(27) and LysRS (28). The exact populations of the open and increases the Asp/Asn discrimination further. In eukaryotic
closedloopstates,withandwithoutboundAsp,areunknown. AspRSs, His-448 is absent, being replaced by an Arg that is
AnotherdifficultyisthatthesubstratesAspandATPareboth moredistantfromtheligandsite.Intheseorganisms,theroleof
charged. Therefore, both short and long range electrostatic ATPasamobilediscriminatoristhereforeimportant,protect-
interactions are expected to play a role in both binding and ingagainstAsnbinding.Inthesamestudy,thecomputational
specificity.Aminoacidbindingmightcoupletoprotonbinding modelwastestedandvalidatedbyexperimentalmeasurements
orreleasebyHis-448orHis-449andtoMg2(cid:1)bindingorrelease ofAsp-stimulatedpyrophosphateexchangeanditsinhibition
byATP. byAsn(36).
AspRSspecificityhasbeenanalyzedwithapowerfulcombi- The present article focuses on the precise stability of the
nation of crystallography, site-directed mutagenesis, kinetic ATP-associatedMg2(cid:1)cationsintheAspRSstructureandtheir
and thermodynamic experiments, and phylogenetic analyses. roleinthethermodynamicsofAspandAsnbinding.Wecon-
Thesemethodshavelimitations,however.Conservedresidues sider AspRS from both E.coli and the archaebacterium Pyro-
maycontributetobinding,bindingspecificity,catalysis,orall coccuskodakaraensis.Wereportfreeenergysimulationsthat
three. Experimental assays based on catalytic activity (29, 30) compare Asp and Asn binding to AspRS in the presence of
usually become infeasible once a single essential residue is either bound ATP(cid:2)Mg2(cid:1) or bound ATP(cid:2)Mg2(cid:1). The level of
2 3
mutated.Thestrengthofelectrostaticinteractionsisverydiffi- Asp/Asndiscriminationineachcasewascomputedusingtwo
cult to infer from crystal structures, because the complex distinct, largely independent methods for the free energy
dielectric environment within a solvated protein causes large changes. The first method, MDFE, alchemically transforms
deviationsfromasimpleCoulomb’slaw(31).Crystallography Asp into Asn during a series of molecular simulations with
doesnotrevealtheionizationstatesofacidicandbasicresidues, anexplicitsolventrepresentation(33).Thesecond,Poisson-
and pK measurements are difficult for AspRS, which is a Boltzmann free energies (PBFE), models the ligand binding
a
homodimerof1180residues.Mostimportantly,weaklypopu- reactionsusingacontinuumdielectricmodelofbothprotein
AUGUST18,2006•VOLUME281•NUMBER33 JOURNALOFBIOLOGICALCHEMISTRY 23793
CationBindingandAspRSSpecificity
andsolvent(35).MDFEwasthenusedtocomputetherelative DataBankentry1IL2;seeabove)andgeneratedsolvated24-Å
affinitiesofAspRSforATP(cid:2)Mg2(cid:1)andATP(cid:2)Mg2(cid:1)andtodem- spherescenteredontheligand.WethenusedpK calculations
2 3 a
onstrate that the ATP(cid:2)Mg2(cid:1) complex has a negligible occu- (see below) to determine the protonation state of His-223, a
2
pancyanddoesnotplayanyroleinthespecificity.Intheclosely histidineresidueorientedintotheATPbindingpocket.LysRS
homologousE.coliLysRS,theATP3-cationcomplexisagain structures were generated from a 2.1-Å resolution E.coli
strongly favored. Binding of the positively charged Lys sub- LysRS(cid:2)Lys(cid:2)ATP(cid:2)Mn2(cid:1) crystal structure (28). We took a 24-Å
3
strate,however,isnotaffectedbycationbinding. spherecenteredonthe(cid:3)-carbonoftheLysligand,replacedthe
We also considered the post-adenylation, AspRS(cid:2)tRNA(cid:2) Mn2(cid:1)cationswithMg2(cid:1),andthensolvatedthesystem.Histi-
AspAMP complex. Our simulations predict a strongly bound dine protonation states were assigned by visual inspection
Mg2(cid:1) cation that aids AspAMP recognition. We show that exceptforHis-270,whichpointsintotheLysRSATP-binding
specificityismaintainedinthepost-adenylationstate(11),with pocket.InbothAspRSandLysRS,theprotonationstateofthe
AspAMPbindingmorestronglythanAsnAMPinthepresence histidine oriented into the ATP pocket is coupled to cation
of one Mg2(cid:1) cation and tRNAAsp. The Mg2(cid:1) cation boosts binding.
AspAMPbindingspecificity,inalongrangeelectrostaticeffect Structures for AspRS with bound AspAMP and tRNA
similar to that of ATP(cid:2)Mg2(cid:1) in the preadenylation complex. were generated from the 2.4-Å resolution E.coli crystal
3
Thus,theintroductionofnegativechargeintoAspRS,bycon- structure 1C0A (18). Again, we considered a 24-Å sphere
formationalchange(36)ortRNAbinding,iscompensatedby centered on the ligand (cid:3)-carbon and solvated the system.
histidine protonation (36) and/or cation binding to preserve pK calculationswereusedtodeterminethepredominantstate
a
Asprecognition. of His-448, a histidine residue close to AspAMP. The cation
Finally, in the supplemental material we have reported a associatedwiththeAspAMPligandphosphategroup(15)was
survey of 238 x-ray structures of protein complexes with placedbystructuralalignmentwiththeprincipalcationinthe
ATPorGTPtakenfromtheProteinDataBank,whichsheds Asp(cid:2)ATP(cid:2)Mg2(cid:1) state (17). The cation occupies the space
3
additionallightontheroleoftheMg2(cid:1)cationsandsupports assignedtowatermolecule1073inthex-raystructure(18)and
thepredicted3-cationAspRSstate.Indeed,wefindthatATP remainsinthesameoctahedralbindingmodethroughoutthe
bindinginacompletelybentconformationwiththreeasso- simulations, coordinating the Glu-482 and Asp-475 carboxy-
ciated Mg2(cid:1) cations is a characteristic property of class II latesandtwoorthreewatermoleculesandremaining4–5Å
aaRSs. awayfromtheAspAMPphosphategroup.
ForbothAspRSandLysRS,iftheentireaaRSproteinwere
EXPERIMENTALPROCEDURES included in the model, rather than a spherical subset, a far
greater number of solvating waters would be needed. To
MolecularDynamicsSimulations
reduce artifacts due to the protein truncation, protein
StartingstructuresforAspRSwithboundAspandATPwere groupsbetween20and24Åfromthecenterwereharmon-
generated from a 2.6-Å resolution crystal structure of E.coli icallyrestrainedtotheirpositionsinthecrystalstructure.In
AspRSwithboundaspartyl-adenylate,AspAMP(ProteinData this way, protein regions beyond 24 Å are accounted for
Bank entry 1IL2) (23). The two tRNA ligands were removed. structurally.Theywillalsobeaccountedforthermodynam-
Weconsideredproteinresidueswithina24-Åspherecentered ically,inaseparatestep(seebelow),wherethefreeenergyto
onthe(cid:3)-carbonoftheadenylateligandofonemonomerofthe reintroducethemissingproteingroupsiscomputedfroma
1IL2 dimer. We overlaid either Asp or Asn on the adenylate continuumelectrostaticmodel.Thishybrid,atomic/contin-
moleculeanddeletedtheoriginalligand.ATPanditsassociated uumapproachisaccurate,becauseearlierworkonthissys-
cations were positioned by taking ATP(cid:2)Mg2(cid:1) from the 1.9-Å temshowedthatbothstructuresandfreeenergiesobtained
3
resolutionP.kodakaraensisAspRS(cid:2)ATPcomplex(ProteinData with spherical subsets of 20-, 24-, or 28-Å radii were all
Bankentry1B8A)(17)andbuildingitintothe1IL2structureso similar(37).
as to overlap with the AMP moiety of the original AspAMP Alllongrangeelectrostaticinteractionswerecomputedeffi-
ligand. Hydrogens were constructed with ideal stereochemis- cientlybytheparticlemeshEwald(PME)method.Foursodium
try. Protonation states of histidines were assigned by visual counterionswereincludedtoreducetheformalchargeofthe
inspection, except for His-448 and His-449 in the active site, system.Onenanosecondofunrestrainedmoleculardynamics
whichwereassignedearlierthroughextensivesimulations(36). wasperformed(foreachcomplex)atconstantroomtempera-
OrientationsofHis,Asn,andGlnsidechainsintheactivesite tureandpressurewithaNose´-Hooveralgorithmfollowing200
weretakenfromthecrystalstructureandverifiedbyinspection ps of thermalization. The complexes modeled are E.coli (23)
(33).Inadditiontocrystalwaters,a73-Åcubicboxofwaterwas AspRS(cid:2)Asp(cid:2)ATP(cid:2)Mg2(cid:1), AspRS(cid:2)Asp(cid:2)ATP(cid:2)Mg2(cid:1), AspRS(cid:2)Asn(cid:2)
3 2
overlaid, and waters overlapping the protein were removed. ATP(cid:2)Mg2(cid:1), and AspRS(cid:2)AsnATP(cid:2)Mg2(cid:1). 500-ps trajectories
3 2
ThefinalmodelcontainedtheaminoacidandATPligands,357 werealsoproducedforeachaminoacidligandinsolution,Asp
proteinresidues,andaround10,000waters,including113crys- orAsn,solvatedatthecenterofaboxofwatermolecules,with
talwaters.Periodicboundaryconditionswereassumed;i.e.the theligand(cid:3)-carbonweaklyrestrainedattheoriginthroughout
entire73-Åboxwasreplicatedperiodicallyinalldirections. thedynamics.
We also generated AspRS(cid:2)Asp(cid:2)ATP complexes from the Additional simulations were performed with a less expen-
P.kodakaraensis AspRS(cid:2)ATP complex (17). We built in Asp sive,spherical,continuumreactionfield(CRF)(37,38)model.
andAsnligandsbyalignmentwiththeE.colistructure(Protein It included the same protein residues as mentioned above,
23794 JOURNALOFBIOLOGICALCHEMISTRY VOLUME281•NUMBER33•AUGUST18,2006
CationBindingandAspRSSpecificity
alongwiththewatermoleculesinsidethe24-Åsphere(about culations were performed for multiple structures, sampled
560waters).Waterandproteinoutsidethe24-Åspherewere every4psalongtheequilibrated3nstrajectory,foratotalof
treated as a single, homogeneous, dielectric medium with a 750structures.Theseparateligandandproteinstructureswere
dielectric constant of 80 (38). Electrostatic interactions be- obtained by simply discarding the unwanted partner. Thus,
tween atoms within the sphere were computed without any structuralrelaxationonseparatingproteinandligandwasnot
cutoff, using an efficient multipole approximation for distant explicitlyincluded(thoughitisimplicitinthedielectriccon-
groups(39).Amultipolarexpansionwith20termswasusedto stant).Thesolventdielectricconstantwassetto80.Thesolute
approximatethereactionfieldduetothesurroundingcontin- dielectricconstantwassetto4,basedonextensiveearliercom-
uum(37,38).Newtoniandynamicswereusedfortheinner20Å parisons between MDFE and PBFE calculations for AspRS
of the sphere and Langevin dynamics for the outer region (35). Proteins groups outside the 24-Å sphere (above) were
(20–24Å),withabathtemperatureof293K.The same four neglected.Thiswasaconvenientandharmlessapproximation,
complexesasaboveweresimulatedfor3nseach,aswellas becausetheyhavebeenshowntocontributelessthan1kcal/
the following 10 complexes (3 ns each): AspRS(cid:2)Asp (or Asn)(cid:2) moltotheAsp/Asnbindingfreeenergydifference(37).Wealso
ATP(cid:2)Mg2(cid:1)(orMg2(cid:1))complexesfromP.kodakaraensis(17);and used PBFE to estimate the binding strengths of each Mg2(cid:1)
3 2
E.coli(28,18)LysRS(cid:2)Lys(cid:2)ATP(cid:2)Mg2(cid:1)(orMg2(cid:1)),AspRS(cid:2)AspAMP cation in AspRS(cid:2)ATP and LysRS(cid:2)ATP, Lys binding to LysRS,
3 2
(or AsnAMP)(cid:2)Mg2(cid:1)(cid:2)tRNA, and AspRS(cid:2)AspAMP (or AsnAMP)(cid:2) AspAMP/AsnAMPbindingtoAspRS(cid:2)tRNA,andcationbind-
tRNAcomplexes.TheCHARMM22forcefield(40)wasusedfor ingtoAspRS(cid:2)aaAMP(cid:2)tRNA,withthesamesetup.
theprotein,ligands,andcounterions.AslightlymodifiedTIP3P MDFE Simulations—Asp/Asn mutation runs were per-
modelwasusedforthewater(41).WeusedtheCHARMMpro- formedwiththePMEsimulationsetup.Theligandside-chain
gram(42),versionc30b1,forallcalculations. geometry, atom types, and charges were reversibly changed
fromtheAspvaluestotheAsnvalues(33–36)overaseriesof
FreeEnergyCalculations
ten100-pssimulationsor“windows.”
The methods used to compute ligand binding free energy HavingidentifiedthemostweaklyboundcationfromPBFE
differencesinAspRShavebeendescribedpreviously(33,36,43, calculations(seeabove),wealsoperformedMDFEsimulations
44). For Asp/Asn binding, we use the thermodynamic cycle to determine the stability of the ATP(cid:2)3-cation complex in
showninFig.1.WeusethehorizontallegsforsimplifiedPBFE AspRS and LysRS, reversibly changing the third cation into a
calculationsandtheverticallegsformorerigorous,alchemical, watermoleculeoveraseriesofMDwindows.Thisprocedure
MDFE simulations. In the latter, the ligand Asp is reversibly was analogous to that used for the Asp/Asn mutations. The
transformedintoAsnduringaseriesofsimulations;thecorre- energyfunctioncanbeexpressedasalinearmixtureofMg2(cid:1)
sponding work is derived from a thermodynamic integration andwaterterms,
formula(43).Similarly,totestthestabilityoftheATP(cid:2)3-cation
complex,themostweaklyboundcation(seebelow)wasrevers- U(cid:2)(cid:4)(cid:3)(cid:5)U0(cid:6)(cid:2)1(cid:7)(cid:4)(cid:3)Uwater(cid:6)(cid:4)UMg2(cid:1) (Eq.1)
ibly transformed into a water molecule (as shown in Fig. 2).
where(cid:4)isaweightor“couplingparameter”andU represents
Related simulations, using the Poisson-Boltzmann linear 0
interactionsbetweenpartsofthesystemotherthanthehybrid
responseapproximation(PB/LRA)method,wereusedtoiden-
ligand.WegraduallymutatedMg2(cid:1)intowaterbychanging(cid:4)
tifythepK ,andhencethepredominantstate,ofproteinhisti-
a from1tozero.Thesuccessiveweightswere(cid:4)(cid:4)0.99,0.95,0.9,
dineresiduesimportantforligandbinding.Seetworecentstud-
0.8,0.6,0.4,0.2,0.1,0.05,and0.01forMg2(cid:1)and1-(cid:4)forwater.
ies(36,45)fordetailsofthemethod.
Thederivativeofthefreeenergy,G,canbewritten,
PBFE Computations—The electrostatic contribution to the
ltirgoasntadtbicinfrdeinegefnreeregeynienrgtyhewalisgoanbdta(cid:2)pinreodtebinyscuobmtrpalcetxinagntdheineltehce- dG/d(cid:4)(cid:5)(cid:5)UMg2(cid:1)(cid:7)Uwater(cid:6)(cid:4) (Eq.2)
separate ligand and protein (35). Non-electrostatic contribu- wherethebracketsrepresentatimeaverageoveranMDtrajec-
tionstothefreeenergywereassumedtocancel.Thisshouldbe toryperformedwiththeenergyfunctionU((cid:4)).Thefreeenergy
agoodapproximationfortheAsp/Asndifferences,becausethe derivativeswerecomputedateach(cid:4)valuefroma100-psMD
twoligandshavethesamesizeandbindinthesameposition simulation.Eachrunthuscorrespondsto1.0nsintotal.
(35). The electrostatic potential was obtained by numerically Equation2canbeusedtoperformagroupdecompositionof
solvingthePoisson-Boltzmannequationusingacubicgridand thefreeenergies(43,46).Indeed,thetermUMg2(cid:1)isasumover
afinitedifferencealgorithm,implementedinCHARMM(grid interactionsbetweenthecationandsurroundingaminoacids
size,144Å;gridspacing,0.4Å).Theprotein(cid:2)solventboundary orwaters,asissimilarlytrueforU .Thus,thefreeenergy
water
wasdefinedbythemolecularsurfaceasinanearlierwork(35). derivative and, ultimately, the free energy can be viewed as a
PBFEwasperformedatzeroionicstrength.Inanearlierstudy sumofgroupcontributions.Suchdecompositionshaveproven
ofAspRS(35),theAsp/Asndiscriminationcomputedwithzero usefulforidentifyingthesourcesofbindingaffinity(44).
and physiological ionic strength differed by just 0.3 kcal/mol. CorrectionforDistantPartsoftheProtein—IntheMDsimu-
Thestructureoftheprotein(cid:2)ligandcomplexwastakenfromthe lations above, protein groups outside of the 24-Å spherical
MDsimulations(above).Giventhelongersamplingtimespos- region were only taken into account structurally through the
sible with CRF and the close similarity between the CRF and applicationofharmonicrestraintstoproteingroupsnearthe
PMEtrajectoriesintermsofbindingpocketgeometryandflex- 24-Åboundary.Theirdirectcontributiontothefreeenergycan
ibility,wegenerallyusedstructuresfromCRFsimulations.Cal- becomputedinasecondstep,wheretheyarereversiblyintro-
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CationBindingandAspRSSpecificity
ducedbackintothesystem.Thefreeenergyforthisstepcanbe threedependingonthestrengthoftheM1/(cid:2)-phosphateinter-
obtainedusingacontinuummodel(asdetailedinRefs37and action,completetheM1coordinationsphere.Oneoftheseisa
47).Acontinuummodelisappropriatebecausethegroupsare highly ordered water, always bridging M1 and Glu-482 in an
morethan24Åawayfromtheligands.Refs.37and47showthat interaction secondary to the direct M1-Glu-482 stabilization.
thenetcontributionofthissecondsteptotheAsp/Asnbinding Anothercation,M2,ispositionedbetweentheATP(cid:2)-,(cid:3)-phos-
freeenergydifferenceislessthan1kcal/mol;thisislessthanthe phates and Glu-482, with three waters also coordinated. M2
overalluncertaintyofbothMDFEandPBFE.Therefore,inthis andGlu-482alsohaveasecondarywater-mediatedinteraction,
work,wedidnotactuallycomputethelongrangecorrection. althoughherethewaterisonlypresent(cid:7)50%ofthetime,with
Wesimplyapproximateditbyzeroandincludeditseffectinthe exchangeinwatermoleculeseveryfewhundredps.Theionized
overalluncertaintyestimate.Notethatinthiswork,continuum residuesAsp-475andGlu-482arehighlyconservedinclassII
electrostaticsareusedinthreedistinctways,whichshouldnot aaRSs; mutagenesis experiments indicate that both are func-
beconfusedwitheachother:1)inthissecond,freeenergystep; tionallyirreplaceableinAspRS(24).
2)forPBFEcalculations(seeabove);and3)intheMDFEsim- Thethirdcation,M3,alsobridgestheATP(cid:2)-,(cid:3)-phosphates
ulationswithCRFboundaryconditions. butdoesnotbindtoprotein.M3remainsonthemoresolvent-
exposedsideofthebindingpocket,completingitsoctahedral
RESULTS
coordinationspherewithfourwatermolecules.Thecoordinat-
Wewillfirstsummarizethemostimportantstructuraland ingwatershaveanaverager.m.s.fluctuationofjust0.4Å.The
dynamic features of the amino acid, ATP, and tRNA binding r.m.s. fluctuations of each of the cations are: M1 (cid:4) 0.24 Å;
sitesinAspRS.Next,wewilldescribeourcalculationstocom- M2(cid:4)0.26Å;M3 (cid:4)0.32Å.Thus,M3ispredictedtohavea
pareAspandAsnbindingtoAspRSinthepresenceofeither crystallographic B-factor that is larger than M1 and M2, in
ATP(cid:2)Mg2(cid:1) or ATP(cid:2)Mg2(cid:1) and calculations to compare agreementwiththeAspRS(cid:2)ATPcrystalstructure(17).M3has
3 2
ATP(cid:2)Mg2(cid:1)andATP(cid:2)Mg2(cid:1)bindingtobothAspRSandLysRS. (cid:7)55watermoleculeswithina9-Åsphere,comparedwith40
3 2
Finally, we report the calculations to compare AspAMP and for the more strongly bound cations, 25 for the amino acid
AsnAMPbindingtoAspRS(cid:2)tRNAinthepresenceofoneorno ligand backbone ammonium group, and 100 for bulk water.
co-boundMg2(cid:1)cations. Finally,theATP(cid:3)-phosphateoxygennotpointingdirectlyto
eitherM2orM3haswater-mediatedinteractionswithM2and
Structure,Dynamics,andSolvationoftheAminoAcid,ATP,
M3throughthewatermoleculescompletingthecationcoordi-
andtRNABindingSitesinAspRS
nationspheres.ForM2,thereisanexchangeinwatermolecules
ThefirstsetofsimulationsweredoneforbothAspandAsn onthesub-100-psscale,sothatonlythewaterassociatedwith
boundtoAspRS,co-boundwithATPandeitherthreeortwo M1andGlu-482aboveandthefourwaterscoordinatedtoM3
cations.TheCRFandPMEmethodsyieldverysimilarbinding aretrulyordered.
pocket structures and dynamics. For the AspRS(cid:2)Asp(cid:2) WhenAsnreplacesAspintheaminoacidbindingsite,earlier
ATP(cid:2)Mg2(cid:1) complex, the r.m.s. deviations from the starting freeenergysimulationsshowedthatHis-448losesitslabilepro-
3
crystal geometry were 0.7 and 1.4 Å for backbone and side- tonandbecomesneutral(36).Inotherrespects,thestructureis
chain atoms, respectively. Fig. 3 illustrates the active site verysimilartotheAspcomplex.LifetimesofH-bondsstabiliz-
dynamicsinatypicalseriesofsnapshotsfromtheMDtrajec- ingtheAsnbackbonegroupsarereducedbyaround10%com-
tory. Fig. 3 was prepared using Molscript (48) and rendered paredwiththeAspcomplex,whereastheAsnside-chaincar-
using Raster3D (49). The cognate Asp ligand makes a stable bonylbindsstronglytoArg-489butnottoLys-198.Overall,the
networkofhydrogenbondstoseveralbindingpocketresidues, meannumberofH-bondsbetweentheaminoacidandAspRSis
whichagreeverywellwiththeavailablecrystalstructures(17, reducedfrom10(Aspcomplex)to7(Asncomplex).
18,23)andpreviousMDsimulations(33–35).FromearlierpK WhenthethirdcationintheATP3-cationcomplex(M3)is
a
calculations(45),His-448isdoublyprotonated(36)andformsa removed,neitherATPnoritsbindingpocketundergoessignif-
saltbridge to the Asp side-chain carboxylate. The Asp side icant displacements. The r.m.s. deviations of ATP and its
chain and backbone amino group are also stabilized by immediateenvironmentfromthestartinggeometryare1.7and
H-bondstoArg-489,Lys-198,aburiedwatermoleculeW1, 1.1Å,respectively,similartothoseseenforthe3-cationcom-
Glu-171oftheclosedflippingloop,andGln-195.Thetermi- plexabove(1.4and1.2Å,respectively).Also,theterminaloxy-
nalcarboxylateformsH-bondswithArg-217and,70%ofthe gens of the aa ligand remain within 3.5–4.9 Å of the ATP
time,withGln-231. (cid:1)-phosphorusinboththe2-cationand3-cationsystems,simi-
ATPretaineditsfullybentgeometry,characteristicofATP lartowhatwasobservedinHisRSsimulations(51).Overall,the
bindingtoclassIIaaRSs(50),throughoutthesimulations.The MDstructuresofthe2-and3-cationcomplexeshaveacompa-
activesitedynamicsshowninFig.3arefromtheE.colistruc- rableagreementwiththeavailablex-raystructures.Thiscon-
ture (23), with ATP(cid:2)Mg2(cid:1) built in from the P.kodakaraensis firms that in the AspRS(cid:2)ATP crystal structure, if a 2-cation
3
structure(17)asdescribedaboveunder“ExperimentalProce- complexwerepresentin10or20%oftheunitcells,itwouldnot
dures.”HerewefocusonthedynamicsoftheATPbindingsite have a noticeable effect on the observed electron density and
in the P.kodakaraensis AspRS(cid:2)ATP complex. The principal couldnotbedetected.Therefore,todeterminetheprobability
Mg2(cid:1)cationnearesttheaminoacid(labeledM1inTable4)is ofa2-cationcomplex,structuraldataisnotenough;freeener-
stabilizedbyH-bondstotheATP(cid:1)-and(cid:2)-phosphatesandalso giesmustbecomputed.Finally,wenotethatthepK calcula-
a
toAsp-475andGlu-482.Twowatermolecules,oroccasionally tions (45) reported in Table 1 indicate that cation binding is
23796 JOURNALOFBIOLOGICALCHEMISTRY VOLUME281•NUMBER33•AUGUST18,2006
CationBindingandAspRSSpecificity
TABLE1
ComputedprotonationstatesforthehistidinenexttothethirdMg2(cid:1)cationintheAspRS(cid:2)Asp(cid:2)ATP,AspRS(cid:2)Asn(cid:2)ATP,andLysRS(cid:2)Lys(cid:2)ATP
complexes:His-223inP.kodakaraensisAspRSandHis-270inE.coliLysRS
ThefinaltwoentriesareforE.coliAspRS(cid:2)aaAMP(cid:2)Mg2(cid:1)(cid:2)tRNAAsp,andcorrespondtoHis-448,closetotheadenylateligandandtheMg2(cid:1)cation.
Complex (cid:2)G (cid:2)G (cid:2)(cid:2)Ga pK shift Hisstate
sol prot a
AspRS(cid:2)Asp(cid:2)ATP(cid:2)Mg2(cid:1) (cid:10)10.6 (cid:10)6.0 (cid:1)4.6(2.4) (cid:1)3.4 Neutral
AspRS(cid:2)Asp(cid:2)ATP(cid:2)Mg32(cid:1) (cid:10)10.6 (cid:10)20.4 (cid:10)9.8(2.4) (cid:10)7.2 Charged
AspRS(cid:2)Asn(cid:2)ATP(cid:2)Mg22(cid:1) (cid:10)10.6 (cid:10)4.1 (cid:1)6.3(2.2) (cid:1)4.6 Neutral
AspRS(cid:2)Asn(cid:2)ATP(cid:2)Mg32(cid:1) (cid:10)10.6 (cid:10)20.9 (cid:10)10.3(2.4) (cid:10)7.5 Charged
LysRS(cid:2)Lys(cid:2)ATP(cid:2)Mg2(cid:1)2 (cid:10)10.6 (cid:1)4.9 (cid:1)15.5(1.9) (cid:1)11.4 Neutral
LysRS(cid:2)Lys(cid:2)ATP(cid:2)Mg32(cid:1) (cid:10)10.6 (cid:10)11.7 (cid:10)1.1(2.2) (cid:10)0.8 Charged/neutral
AspRS(cid:2)AspAMP(cid:2)M2g2(cid:1)(cid:2)tRNAAsp (cid:10)10.6 (cid:10)19.1 (cid:10)8.5(1.6) (cid:10)6.2 Charged
AspRS(cid:2)AsnAMP(cid:2)Mg2(cid:1)(cid:2)tRNAAsp (cid:10)10.6 (cid:10)7.5 (cid:1)3.1(1.3) (cid:1)2.3 Neutral/charged
a(cid:8)(cid:8)G(cid:4)(cid:8)G (cid:10)(cid:8)G (inkcal/mol;seeRefs.36and45formoredetails)(sol,solvent;prot,protein).Uncertainty,showninparentheses,isestimatedfromthedeviationin
computed(cid:8)prGot valsuoels,samplingevery4psalong0.5-nssegments.
prot
coupledtonearbyhistidinecharging.TheATPbindingpockets TABLE2
ofbothAspRSandLysRSfeatureahistidineresiduepointing MDFEandPBFEbindingfreeenergydifferences(cid:2)(cid:2)G(kcal/mol)for
toward the ATP (cid:3)-phosphate (17, 28). The structure and AATspP(cid:2)vMegrs2u(cid:1)sAsninAspRS,aloneandwithco-boundATP(cid:2)Mg32(cid:1)and
2
dynamics of the LysRS substrate binding pocket will be PBFE(cid:8)(cid:8)GvaluesarealsogivenforAspAMPversusAsnAMPbindinginAspRS,
described in detail elsewhere. This histidine is neutral in the withtRNAAspandeitheroneorzeroco-boundcations.MDFE(cid:8)(cid:8)Giscomputed
fromthevertical,alchemicallegsinFig.1:(cid:8)(cid:8)G(cid:4)(cid:8)G (cid:10)(cid:8)G.Apositivesign
presence of ATP(cid:2)Mg32(cid:1) but can gain a positive charge when correspondstopreferentialAspbinding.(cid:8)G1and(cid:8)G4a1recomp4utedbyalchemi-
ATP(cid:2)Mg2(cid:1) is artificially changed to ATP(cid:2)Mg2(cid:1) (Table 1). As callytransformingAspintoAsn(orthereverse)duringaseriesofsimulations.The
3 2 MDFE(cid:8)(cid:8)Gvaluesareaveragesoverfour1.0-nsruns;PBFE(cid:8)(cid:8)Gvaluesarecom-
shownintheenergeticanalysisbelow,thishistidinecharging putedfrom3nsnativeMDtrajectories,usingthehorizontal,bindinglegsinFig.1.
doesnotfullycompensateforthecostofunbindingthethird AspRS(cid:1)indicatesadoublyprotonatedHis-448.
cation,sothatthestatewiththreecationsisstronglyfavored Medium MDFE(cid:2)(cid:2)G PBFE(cid:2)(cid:2)G
(Asp3Asn)a (Asp3Asn)b
andalwayspresent.
AspRS (cid:1)3(2)
FollowingourextensiveanalysisofAspRSsubstratespecific- AspRS(cid:1) (cid:1)11(2) (cid:1)10(2)
ityinthepreadenylationstep(presentworkandRefs.33–36), AAssppRRSS(cid:1)(cid:2)A(cid:2)ATTP(cid:2)PM(cid:2)Mg32g(cid:1)2(cid:1) (cid:1)(cid:1)199((33)) (cid:1)18(2)
the last system we consider here involves AspAMP/AsnAMP AspRS(cid:2)ATP(cid:2)Mg2(cid:1)3 (cid:1)5(1) (cid:1)5(2)
specificityinthepresenceofco-boundtRNAinthepost-adeny- AspRS(cid:1)(cid:2)aaAMP2(cid:2)Mg2(cid:1)(cid:2)tRNAAsp (cid:1)9(2)
AspRS(cid:1)(cid:2)aaAMP(cid:2)tRNAAsp (cid:1)4(3)
lationstep.Openingofthe“flippingloop”accompaniestRNA
AspRS(cid:2)aaAMP(cid:2)Mg2(cid:1)(cid:2)tRNAAsp (cid:1)4(2)
binding,asshowninthex-raystructure(18).Apartfromthe
aMDFEvaluesarefromRef.36.Uncertainty(showninparentheses)inMDFEdata
lossoftheGlu-171interaction,thestructureanddynamicsof isobtainedbyfirstaveragingeachpairofforward/backwardrunsandthentaking
twicethedeviationamongpairs.Theestimateduncertaintyismuchsmallerthan
the AspAMP binding site are similar to those in the
thedifference(or“hysteresis”)betweenforwardandbackwardruns(seetext).
Asp(cid:2)ATP(cid:2)AspRS systems described above, in agreement with bUncertaintyinPBFEdataisestimatedfromthedeviationincomputedPBFE(cid:8)(cid:8)G
values,samplingevery4psalong1.0–3.0-nssegments.
thex-raydata(17).Ther.m.s.deviationsofheavyatomsfrom
thestartinggeometryinAspAMPanditsimmediateenviron-
mentare0.7and1.0Å,respectively.AsnAMPisslightlymore Asp/AsnDiscriminationbyAspRSinthePresenceofATPand
mobilethanAspAMPinthebindingpocket.Ther.m.s.devia- EitherThreeorTwoMg2(cid:1)Cations
tions of AsnAMP and its immediate environment from the
WefirstcomparedthebindingfreeenergiesofAspandAsn
startinggeometryareboth1.1Å.AsshowninTable1,His-448
inthepresenceofATPwiththreeassociatedcations.Wecom-
losesitsextraprotonandbecomesneutralinthepresenceof
paredMDFEresults(someofthemobtainedearlier(36))with
AsnAMPandaco-boundMg2(cid:1)cation.Thisdoesnotleadto
the PBFE results obtained here. The data are summarized in
significant changes in ligand or binding site structure; His- Table2.EachMDFE(cid:8)(cid:8)Gvaluewascomputedfromtwosolu-
448simplyswingsawayfromAsnAMPtowardthesolvent- tion simulations and four protein simulations (of ten 100-ps
exposedsideoftheAspRSpocket.TheMg2(cid:1)cationassoci- windowseach;see“ExperimentalProcedures”).Insolution,one
ated with the aaAMP phosphate maintains a strong runwasperformedineachdirection,mutatingAsptoAsnor
coordination with Glu-482 and Asp-475 throughout the thereverse,yielding(cid:8)G (seeFig.1).Intheprotein,tworuns
1
dynamicsandhasanr.m.s.fluctuationofonly0.30Å,similar wereperformedineachdirection,yielding(cid:8)G (Fig.1).When
4
tothecationscoordinatingATPinthepreadenylationstep ATP(cid:2)Mg2(cid:1)isco-bound,(cid:8)(cid:8)G(cid:4)(cid:1)19kcal/mol,favoringAsp.
3
(above). The co-bound tRNAAsp molecule does not undergo This decreases to (cid:1)9 kcal/mol when His-448 is artificially
significant deviations from its starting geometry. The r.m.s. maintainedinthesinglyprotonatedstate(36).NoticethatHis-
deviation of the tRNA from its x-ray position is 1.4 Å, with 448isabsentfrommosteukaryoticAspRSs,beingreplacedby
either AspAMP or AsnAMP co-bound. Finally, removing the an Arg that is more distant from the ligand site. PBFE free
Mg2(cid:1) cation associated with aaAMP causes only a slight energyvaluesareobtainedfromtheverticallegsofthethermo-
increase in structural disorder. The r.m.s. deviations for dynamiccycleinFig.1.Inthethreemostimportantstatesfor
AspAMP, tRNA, and their immediate environments increase whichbothMDFEandPBFEsimulationswereperformed,the
by0.1–0.2Åwhenthecationisartificiallyremoved. PBFEestimatesmatchtheMDFEvaluestowithin(cid:9)2kcal/mol.
AUGUST18,2006•VOLUME281•NUMBER33 JOURNALOFBIOLOGICALCHEMISTRY 23797
CationBindingandAspRSSpecificity
TABLE4
Electrostatic(PBFE)contributiontothebindingfreeenergiesofeach
cationinAspRS(cid:2)ATP(cid:2)Mg2(cid:1)andLysRS(cid:2)ATP(cid:2)Mg2(cid:1)complexesandof
thesingleremainingcat3ioninAspRS(cid:2)aaAMP(cid:2)M3g2(cid:1)(cid:2)tRNAAsp
LysRS(cid:1)denotesLysRSwiththenear-M3His-270positivelycharged,andAspRS(cid:1)
denotesAspRSwiththenear-M1His-448positivelycharged.(cid:8)Gvalueshavestand-
arddeviationsof1–3kcal/molandwerecomputedfromatleast250MDsnapshots
sampledevery4psovermulti-nanosecondMDtrajectories.
Cationbinding(cid:2)G
Complex
M1 M2 M3
kcal/mol
AspRS(cid:2)Asp(cid:2)ATP(cid:2)Mg2(cid:1) (cid:10)104 (cid:10)144 (cid:10)96
AspRS(cid:2)Asn(cid:2)ATP(cid:2)Mg32(cid:1) (cid:10)85 (cid:10)135 (cid:10)81
FIGURE1.Thermodynamiccycleusedforcomputationoftheaminoacid AspRS(cid:2)Asp(cid:2)ATP(cid:2)Mg23(cid:1)a (cid:10)110 (cid:10)138 (cid:10)103
fbaoriernldfirgienaegnedfnrmeereugetyancteihorangnyigndesisfoffloeurrteiAonsncneaasnn.dd(cid:8)eGAn1szpaynbmdine(cid:8),dGrien4sgrpetefoecterinvtoezylfyrm,eaeen.edne(cid:8)rGg2yacnhdan(cid:8)gGes3 ALLyyssspRRRSSS(cid:2)(cid:1)L(cid:2)A(cid:2)yLssyn(cid:2)As(cid:2)(cid:2)AATTTPP(cid:2)PM(cid:2)(cid:2)MMgg32g(cid:1)3322(cid:1)(cid:1)a (cid:10)(cid:10)(cid:10)111032924 (cid:10)(cid:10)(cid:10)111333730 (cid:10)(cid:10)(cid:10)1870483
AspRS(cid:1)(cid:2)AspAMP(cid:2)M3g2(cid:1)(cid:2)tRNAAsp (cid:10)117
TABLE3 AspRS(cid:1)(cid:2)AsnAMP(cid:2)Mg2(cid:1)(cid:2)tRNAAsp (cid:10)110
Electrostatic(PBFE)contributiontothebindingfreeenergiesfor aThestartingstructureistheP.kodakaraensisAspRS(cid:2)ATPcomplex(seeRef.17).
AspRS(cid:2)Asp,LysRS(cid:2)Lys,andAspRS(cid:2)AspAMPcomplexationasa AllotherstartingstructuresarefromE.colicomplexes.
functionofthenumberofboundMg2(cid:1)cations
LysRS(cid:1)denotesLysRSwithHis-270positivelycharged;AspRS(cid:1)isAspRSwith
His-448positivelycharged.(cid:8)Gvalueshavestandarddeviationsof1–3kcal/moland boundcationineachenzyme.Asolutedielectricconstantof4
werecomputedfromatleast250MDsnapshotssampledevery4psovermulti- was used, with structures sampled from the Asp(cid:2)AspRS,
nanosecondMDtrajectories. Asn(cid:2)AspRS,andLys(cid:2)LysRSMDtrajectories.
Complex Aminoacidligandbinding(cid:2)G
FromTable4,itisclearthatM3istheleaststablecationinall
kcal/mol
thecomplexes.InAspRS,theorderofcationbindingstrengths
AspRS(cid:2)Asp(cid:2)ATP(cid:2)Mg2(cid:1) (cid:10)38
AspRS(cid:2)Asp(cid:2)ATP(cid:2)Mg32(cid:1) (cid:10)31 isM2(cid:11)M1(cid:11)M3,irrespectiveoftheaminoacidligand.M3is
LysRS(cid:2)Lys(cid:2)ATP(cid:2)Mg2(cid:1)2 (cid:10)61 onthesolvent-exposedsideofATP.Itsr.m.s.fluctuationsare
LysRS(cid:1)(cid:2)Lys(cid:2)ATP(cid:2)M3g2(cid:1) (cid:10)61
LysRS(cid:2)Lys(cid:2)ATP(cid:2)Mg2(cid:1)3 (cid:10)60 about25%largerthanthoseofM1andM2.Ofthethreecations,
LysRS(cid:1)(cid:2)Lys(cid:2)ATP(cid:2)M2g2(cid:1) (cid:10)59 M1,coordinatingtheATP(cid:1),(cid:2)-phosphatesAsp-475andGlu-
AspRS(cid:1)(cid:2)AspAMP(cid:2)tR2NAAsp (cid:10)35 482, is the most strongly stabilized by Asp rather than Asn
AspRS(cid:1)(cid:2)AspAMP(cid:2)Mg2(cid:1)(cid:2)tRNAAsp (cid:10)48
binding.Itsbindingfreeenergyis19kcal/mollowerwithbound
AspthanwithboundAsn.M1isthecationclosesttotheamino
This is comparable with the uncertainty in the MDFE values acid,andhenceitisthemoststronglyaffectedbythenetcharge
themselves. oftheaminoacidligand.M2isthemosttightlyboundcation,
WhenATPisassociatedwithonlytwocations,theAsp/Asn coordinatingthe(cid:2)-and(cid:3)-phosphatesandGlu-482.M2isfur-
binding free energy difference decreases substantially, to thest from the amino acid binding site, and hence the least
(cid:8)(cid:8)G (cid:4) (cid:1)5 kcal/mol, compared with 9 kcal/mol with affectedbytheaminoacididentity;itsbindingfreeenergyis9
ATP(cid:2)Mg2(cid:1)(Table2).AgreementbetweenMDFEandPBFEis kcal/mollowerwithboundAspthanwithboundAsn.M3,the
3
good.Thus,eventhoughthethirdMg2(cid:1)cationis11Åaway most weakly bound cation, coordinates the (cid:2)- and (cid:3)-phos-
fromthe(cid:3)-carbonoftheligandsidechain,itinteractsstrongly phatesbutnotprotein.M3isintermediateindistancefromthe
withtheaminoacidligandandstronglyfavorsthenegativeAsp aminoacidpocketandintermediateinitsenergetics;itsbind-
overneutralAsn.Consistentwiththisfinding,PBFEestimates ingfreeenergyis15kcal/mollowerwithboundAspthanwith
ofaminoacidbindingstrengthsinTable3showthatremoving bound Asn. This situation can be compared with the LysRS
the third cation strongly reduces Asp binding to AspRS. The case.Table4showsclearlythatinLysRS,M3isagainthemost
results for LysRS are also shown in Table 3. We see that Lys weakly bound cation. M1 and M2 have similar binding
bindingtoLysRSisnotsensitivetothenumberofMg2(cid:1)cations strengths;botharestronglystabilizedbynearbyLysRSgluta-
present,despiteitspositivechargeandincontrasttothelarge materesidues.
effectofthecationsinAspRS.Thisisshownbythenearequality Relative Stabilities of the ATP(cid:2)Mg2(cid:1) and ATP(cid:2)Mg2(cid:1) Com-
3 2
oftheLysbindingfreeenergiesfortheLysRS(cid:2)Lys(cid:2)ATP(cid:2)3-cation plexes in Alchemical MDFE Simulations—ATP co-binding
complex(witheitherneutralorchargedHis-270)andforthe alongwiththreedivalentcationsissupportedbutnotprovedby
2-cationcomplex(witheitherneutralorchargedHis-270).In the AspRS(cid:2)ATP and LysRS(cid:2)ATP crystal structures (17, 28).
thenextsection,weshowthatinbothAspRSandLysRS,ATP Indeed,thecrystallographicdatacannotruleoutapartialoccu-
alwaysbindswithallthreecations. pancyforthethirdcation.Inotherwords,thethirdcationcould
bepresentinfewerthan100%ofthecrystalcells.Fromthedata
StabilityoftheATP(cid:2)Mg2(cid:1)ComplexinAspRSandLysRS
3 presentedabove,apartialoccupancy,evenashighas80or90%,
Identifying the Most Weakly Bound Cation—X-ray crystal wouldhaveasignificanteffectontheAsp/Asndiscrimination
structuresofATPboundtoAspRSandLysRSshowthatboth inAspRS.Therefore,weperformedalchemicalfreeenergysim-
AspRSandLysRSareamongtheclassIIsynthetasesthatpref- ulations in which the third Mg2(cid:1) cation, M3, was reversibly
erentiallybindthreedivalentcations(17,28).PBFEcalculations transformed into a water molecule, both in the enzyme and
reported in Table 4 were used to identify the most weakly aloneinsolution.Thiscationwaschosenbecauseitisthemost
23798 JOURNALOFBIOLOGICALCHEMISTRY VOLUME281•NUMBER33•AUGUST18,2006
CationBindingandAspRSSpecificity
TABLE5
StabilityofthethirdMg2(cid:1)cationinAspRSandLysRSATP(cid:2)3-cation
complexesusingMDFE
Energiesareinkcal/mol.(cid:8)GcorrespondstothemutationofMg2(cid:1)intoawater
molecule.LysRS(cid:1)denotesLysRSwiththenear-M3His-270positivelycharged.
“Forward”runstransformMg2(cid:1)intoawater;“backward”runstransformawater
intoMg2(cid:1).(cid:8)Ginproteiniscomputedforeachsystemfromthefour(cid:8)Gvalues
given,correspondingtorunsfromindependentstartingstructures.Apositive(cid:8)(cid:8)G
FIGURE 2. Thermodynamic cycle for binding of the third cation in correspondstopreferentialMg2(cid:1)bindingtotheenzyme.Uncertainty(shownin
AspRS(cid:2)Asp(cid:2)ATP.Ananalogouscyclewasusedforbindingofthethirdcation parentheses)isobtainedbyfirstaveragingeachpairofforward/backwardrunsand
inLysRS(cid:2)Lys(cid:2)ATP. thentakingtwicethedeviationamongpairs.
weakly bound of the three (Table 4). The data were analyzed Medium Dailrcehcetmioincaolf (Mg2(cid:1)(cid:2)3Gwater) (cid:2)(cid:2)G
mutationrun
withthethermodynamiccycleinFig.2.Thedoublefreeenergy
difference (cid:8)(cid:8)G represents the standard free energy for bind- Solution Forward (cid:1)430
Solution Backward (cid:1)438
ing the third cation to the preformed AspRS(cid:2)aa(cid:2)ATP(cid:2)Mg2(cid:1) AspRS(cid:2)Asp(cid:2)ATP(cid:2)Mg2(cid:1) Forward (cid:1)534/(cid:1)528
complex. 2 ALysspRRSS(cid:2)L(cid:2)Ayssp(cid:2)A(cid:2)ATTPP(cid:2)M(cid:2)Mgg2(cid:1)222(cid:1) BFoarcwkwaradrd (cid:1)(cid:1)458121//(cid:1)(cid:1)550127 (cid:1)78(10)
Table 5 shows the computed free energy changes. We LysRS(cid:2)Lys(cid:2)ATP(cid:2)Mg22(cid:1) Backward (cid:1)452/(cid:1)468 (cid:1)53(8)
obtainedlargedeviationsbetweenforwardandbackwardruns LysRS(cid:1)(cid:2)Lys(cid:2)ATP(cid:2)M2g2(cid:1) Forward (cid:1)500/(cid:1)480
inenzyme(i.e.whereMg2(cid:1)ismutatedintowaterorthereverse; LysRS(cid:1)(cid:2)Lys(cid:2)ATP(cid:2)Mg222(cid:1) Backward (cid:1)465/(cid:1)434 (cid:1)36(16)
third column in Table 5). In fact, previous experience with
chargingfreeenergycalculationsinAspRSandothersystems
lowerthanthestandardstatevalue.Overall,forbothAspand
suggeststhatwhenforwardandbackwardresultsareaveraged,
Asn,thethirdcationismuchmorestableboundtoAspRSthan
there is a significant compensation of errors. For example,
aloneinsolution,andsoATPalwaysbindstoAspRSwithall
when Asp is transformed into Asn in AspRS, there is a large
three associated Mg2(cid:1) cations; the Boltzmann probability of
forward/backward free energy hysteresis, but the forward/
the2-cationstateisinfinitesimal.Thesamequalitativeresultis
backwardaveragechangesbylessthan4kcal/molwhentherun
foundforLysRS;the3-cationcomplexisfavoredby52kcal/mol
lengthisdoubled(seesupplementalmaterial).Thus,estimated
in the standard state and by at least 47 kcal/mol at cellular
statisticaluncertaintyisobtainedbyfirstaveragingpairsoffor-
concentrations.Itiseasytoshow,usingthedatainTables1and
ward/backwardrunsandthentakingtwicethestandarddevia-
tionamongthese.ForMg2(cid:1)bindingtoLysRS(cid:1),thestandard 5,thatstatesinwhichtheaaRS(cid:2)aa(cid:2)ATPcomplexbindsonlyone
errorestimate(16kcal/mol)isabouthalfthecomputed(cid:8)(cid:8)G or no Mg2(cid:1) cations are negligibly populated compared with
eventheaaRS(cid:2)aa(cid:2)ATP(cid:2)Mg2(cid:1)states.NotethatMg2(cid:1)competi-
(36 kcal/mol). Thus, even though the precision of the MDFE 2
dataislow,thepredictedMg2(cid:1)bindingtrendsareverystrong tion with monovalent ions is also insignificant, because the
andcanbetakenasqualitativelyaccurate. most abundant ion, potassium, has a concentration of about
Whenthe third cation binds, the nearby His protonation 150mM.ThestandardbindingfreeenergyofK(cid:1)canbeesti-
statecanchange,asshowninTable1.Tocomparethemost mated from electrostatic considerations to be weak, on the
relevant states with 2 and 3 bound cations, the MDFE data orderofhalfthatofthethirdMg2(cid:1)cation.Thus,mixedcom-
mustbecombinedwiththecomputedprotonationfreeener- plexessuchasATP(cid:2)Mg22(cid:1)(cid:2)K(cid:1)areexpectedtobenegligiblypop-
gies(Table1).Thus,whenthethirdcationbindstoLysRS(cid:1), ulatedinbothAspRSandLysRS.
the nearby His-270 becomes deprotonated, and the free To identify the most important interactions stabilizing the
energyisloweredbyanadditional16kcal/mol(Table1).The thirdcation,weperformedagroupdecompositionofthecom-
overallbindingfreeenergyofthethirdcationtoLysRSisthere- puted binding free energy based on Equation 2 (46, 43). The
fore estimated to be (cid:10)36(cid:10)16 (cid:4) (cid:10)52 kcal/mol, correspond- resultingfreeenergycomponentsaregiveninTable6.Avery
ing to the process Mg2(cid:1) (cid:1) LysRS(cid:1)(cid:2)Lys(cid:2)ATP(cid:2)Mg2(cid:1) 3 large,favorablecontribution((cid:1)367kcal/mol)comesfromthe
LysRS(cid:1)(cid:2)Lys(cid:2)ATP(cid:2)Mg2(cid:1)3LysRS(cid:2)Lys(cid:2)ATP(cid:2)Mg2(cid:1)(cid:1)H(cid:1).N2 otice ATP(cid:2)Mg2(cid:1)moietyitself,eventhoughitiselectricallyneutral.
that the estimated u3ncertainty in the compu3ted protonation Another2(cid:1)126 kcal/mol come from the conserved Glu-482/
freeenergyismoderate(Table1).Similarly,forAspRS,wecon- Asp-475sidechains,eventhoughtheseare6/9Åawayandare
sidertheprocessMg2(cid:1)(cid:1)AspRS(cid:1)(cid:2)Asp(cid:2)ATP(cid:2)Mg2(cid:1)3Mg2(cid:1)(cid:1) primarilyinvolvedincoordinatingtheothertwocations.The
2
AspRS(cid:2)Asp(cid:2)ATP(cid:2)Mg2(cid:1)(cid:1)H(cid:1)3AspRS(cid:2)Asp(cid:2)ATP(cid:2)Mg2(cid:1)(cid:1)H(cid:1). contribution of solvent is small at (cid:10)73 kcal/mol (opposing
2 3
The total binding free energy is (cid:1)10(cid:10)78 (cid:4) (cid:10)68 kcal/mol. binding). The solvent contribution can be interpreted as a
Finally, the four AspRS(cid:2)aa(cid:2)ATP(cid:2)Mg2(cid:1) systems (aa (cid:4) Asp or dielectric shielding of the negative charges that favor Mg2(cid:1)
n
Asn;n(cid:4)2or3)formathermodynamiccycle,allowingustoinfer binding. Other protein groups make much smaller contribu-
also the free energy for binding the third cation to the AspRS(cid:2) tions. For example, the net contribution of the nearby salt
Asn(cid:2)ATP(cid:2)Mg2(cid:1)complex.Giventhatbindingofthethirdcation bridgepair,Glu-219/Arg-225,isjust(cid:1)11kcal/mol,andnearby
2
inAspRS(cid:2)Asp(cid:2)ATPisfavoredby68kcal/mol,thethirdcationin Glu-171 and Arg-217 contribute just (cid:1)14 kcal/mol. The Asp
AspRS(cid:2)Asn(cid:2)ATPisfavoredby59kcal/mol. ligand contributes (cid:1)45 kcal/mol. Overall, M3 binding is
TheconcentrationofMg2(cid:1)inthecellisontheorderof1mM favoredlargelybyATP(cid:2)Mg22(cid:1),theconservedGlu-482/Asp-475
(52).Thefreeenergychangesreportedabovecorrespondtoa pair, and the Asp ligand. On balance, these groups stabilize
standardstateof1MMg2(cid:1).Thus,atphysiologicalMg2(cid:1)con- cation(cid:2)proteinbindingcomparedwithcationsolvationinbulk
centrations, the binding free energy is at most 3–5 kcal/mol water(Tables5and6).
AUGUST18,2006•VOLUME281•NUMBER33 JOURNALOFBIOLOGICALCHEMISTRY 23799
CationBindingandAspRSSpecificity
TABLE6 directly.Nevertheless,severallinesofargumentstronglysug-
FreeenergycomponentsforthestabilityofthethirdMg2(cid:1)cationin gestthatourmainresultsaremeaningful.
theAspRSATP(cid:2)3-cationcomplexusingalchemicalMDFEsimulations
Forthecrucial,longrange,electrostaticinteractions,weused
Energiesareinkcal/mol.(cid:8)GcorrespondstothemutationofMg2(cid:1)intoawater
molecule; total (cid:8)G (cid:4) (cid:1)528 kcal/mol (Table 5), comprising the sum of the two recent, sophisticated methods, including one that com-
ATP(cid:2)Mg22(cid:1),water,Aspligand,andtotalproteincontributions,togetherwith(cid:1)15 bines an atomic representation of groups near the active site
kcal/molforthePoisson-Boltzmanncontinuumcorrection(seeRef.33fordetails)
and(cid:1)2kcal/molforvanderWaalsinteractions.Apositivecomponentindicates withacontinuumrepresentationofdistantgroups(37,47).The
stabilizationofMg2(cid:1)bindingtotheenzyme. simplifiedtreatmentofdistantgroupsisquiteaccurate.Indeed,
Group Component calculationsthatuseatomicregionsofdifferentsizesgivevery
WATaPte(cid:2)Mr g22(cid:1) (cid:1)(cid:10)37637 similar free energy changes. We note that although substrate
Aspligand (cid:1)45 interactions with distant groups have been observed experi-
Asp-475 (cid:1)48 mentally and discussed extensively, they are weak compared
Glu-482 (cid:1)78
Glu-219 (cid:1)32 with the effects of interest here. For example, AspRS is a
Arg-225 (cid:10)21 homodimer, and a certain cooperativity is found for Asp and
Glu-171 (cid:1)37
Arg-217 (cid:10)23 ATPbindingtothetwoactivesites(53).Butthetwobinding
constantsforATPdifferbyafactorofonly10,correspondingto
1.4 kcal/mol of binding free energy; Mg2(cid:1) effects are much
AspAMP/AsnAMPDiscriminationinthePresenceoftRNAand
larger.
EitherOneorZeroMg2(cid:1)Cations
Toobtainthermodynamicinformation,wedidMDFEsimu-
TofurtherprobetheroleofcationsinAspRSsubstratebind-
lations. MDFE has given reasonable accuracy for several pro-
ing specificity, we performed simulations of AspAMP and
teinswheredirectexperimentalcomparisonswerepossible;see
AsnAMP binding to AspRS in the presence of a single Mg2(cid:1)
recentworkonredoxpotentials(54),side-chainpK shifts(55),
cationandtheco-substratetRNA.Thissystemcorrespondsto andprotein(cid:2)proteinbinding(56,57).ItsvalidityforaAspRSwas
thepost-adenylationstep,immediatelybeforetRNAaminoacy-
testedearlierbycomparisonwithexperimentalmeasurements
lation.AMg2(cid:1)cationisgenerallythoughttobeassociatedwith
ofAsp-stimulatedpyrophosphateexchange(36).Wemadesys-
the aaAMP substrate (15), although its position is not always
tematic comparisons with PBFE, an independent method
explicitly determined in x-ray structures. In our MD simula-
that has been extensively calibrated and tested for AspRS,
tions, the cation remained strongly coordinated to the con-
andfoundgoodagreement.Wealsoexploredthesensitivity
served acidic residues Glu-482 and Asp-475 in the binding
of our results to system details by studying several AspRS
pocket.PBFEcationbindingcalculations(Table4)confirmthat
variants:E.coliAspRSwithandwithouttRNAandP.kodak-
theMg2(cid:1)cationisverystronglyheld,withanestimatedelec-
araensis AspRS. Considering the different amino acid
trostatic contribution to the binding free energy of 100 kcal/
ligandsandthenumbersofMg2(cid:1)cationsandHisprotona-
mol.Thisiscomparablewiththetwostrongestcationsinthe
AspRS(cid:2)aa(cid:2)ATP(cid:2)Mg2(cid:1) complexes (Table 4). PBFE calculations tionstates,wesimulated24differentsystemsforover75ns,
3 whichisseveraltimesthetotalsimulationlengthinrelated
reported in Table 2 show that this cation is crucial for
studies(54,55,56).
AspAMP/AsnAMP discrimination. For AspRS with the
Severalpredictionscouldbetestedexperimentally,inprin-
co-bound cation and a positively charged His-448, we com-
ciple.ForLysRS,onecouldmeasuretheeffectofMg2(cid:1)onthe
puted an AspAMP/AsnAMP binding free energy difference
(cid:8)(cid:8)G of (cid:1)12 kcal/mol in favor of AspAMP. pK calculations Lys affinity (predicted here to be small) using an established
a fluorescence assay (58). For both AspRS and LysRS, we pre-
(Table 1) indicate a preference for neutral His-448 when
dictedthatATPisalwaysboundwiththreeMg2(cid:1)cations,so
AsnAMPreplacesAspAMP,witha3kcal/molfreeenergygain
that the rate constant, k , for the chemical adenylation step
whenHis-448becomesneutral.Wethereforesubtractedthis3 cat
kcal/mol(Table1)fromourPBFE(cid:8)(cid:8)G,givingacorrected(cid:8)(cid:8)G should be independent of Mg2(cid:1) concentration. We look for-
of (cid:1)9 kcal/mol in favor of AspAMP (Table 2). Artificially wardtothesepredictionsbeingtestedbyothers.
ComplexityoftheAminoAcidBindingReaction—AspRS(cid:2)aa
removingthecationreducestheAspAMP/AsnAMPdifference
strongly,witha(cid:8)(cid:8)Gofjust(cid:1)4kcal/mol.Interestingly,ifone recognitionisespeciallycomplexbecauseitinvolves“hidden”
keepsthecationbutartificiallyswitchesHis-448toitsneutral reorganization, substrate-assisted specificity, and long range
state,oneobservesthesameneteffect,witha(cid:8)(cid:8)Gofjust(cid:1)4 interactions.Indeed,thebindingpocketislargelypreorganized,
kcal/mol. This offers a further illustration of the coupling and rearrangement upon aa binding is mostly limited to the
betweencationsintheATP/AMPbindingsiteandtheionizable flippingloop,whichbringsitsnegativeGlu-171intothepocket.
His-448,neartheaminoacidsidechain. However, a hidden reorganization also occurs, with His-448
bindingaprotontoopposetheGlu-171charge.Thislabilepro-
DISCUSSION tonfavorsAspbindingandhelpsdiscriminateagainstAsn.
Validation of Simulation Methodology—Despite the power ATP(cid:2)Mg2(cid:1) acts as another mobile discriminator, so that
3
of modern structural biology, molecular recognition is too AspRSspecificityissubstrate-assisted.ThemostdistantMg2(cid:1)
complextobecompletelyunderstoodwithexperimentsalone: cation contributes 4 kcal/mol to the Asp/Asn binding free
crystalstructuresdonotshowelectricfields.MDgivescomple- energydifference.Thestrengthofthecouplingbetweentheaa
mentaryinformationthatisdifficulttoobtainexperimentally. sidechainandthiscationisremarkable,giventhattheyare11Å
Forthesamereason,someofourpredictionsarehardtotest apart. We note that analogous couplings have been observed
23800 JOURNALOFBIOLOGICALCHEMISTRY VOLUME281•NUMBER33•AUGUST18,2006
CationBindingandAspRSSpecificity
M3, respectively. Their negative
charges,alongwiththefournegative
phosphate charges, exactly balance
thechargesoftheMg2(cid:1)cations.A
large, unfavorable contribution
((cid:10)73kcal/mol)arisesfromsolvent,
correspondingtoadielectricshield-
ing of the negative charges in the
binding pocket. Finally, the Asp
ligand itself contributes (cid:1)45 kcal/
mol,anotherexampleoflongrange
couplingintheAspRSactivesite.
Mg2(cid:1)ContributestoAspRSSpec-
ificity in Synergy with Other Active
SiteGroups—Thisandearlierstud-
ies show that AspRS amino acid
specificity arises from a complex
network of electrostatic interac-
tions involving conserved side
FIGURE3.Wall-eyedstereoviewof11snapshotsoftheAspRS(cid:2)Asp(cid:2)ATPactivesiteat20-psintervals,from chains and other factors. We have
thelast200psoftheMDtrajectory.AspandATPareshowninball-and-stickrepresentation.Mg2(cid:1)cations
areshownasyellowspheres.Themostweaklyboundcation,M3,isinfront.Importantbindingpocketresidues focusedhereonATPanditsassoci-
arelabeledandcoloredaccordingtocharge:red,negative;blue,positive;orange,neutral.Astablewater atedcationsfoundtoplayanimpor-
moleculeisshowningreenandlabeledW.Theproteinbackboneisshownintuberepresentationwithflipping
tantrole.Thesimulationsshowthat
loopresiduescoloredgreen.ThisfigurewaspreparedusingMolscript(48)andRaster3D(49).
iftheweakestboundMg2(cid:1)cationis
removed, the Asp/Asn discrimina-
betweenaaandATPbindinginotheraaRSs,e.g.ThrRS(59)and tion is strongly reduced. The binding free energy difference
MetRS(60).Butthecouplingherewassignificantlylarger. (cid:8)(cid:8)G drops from 9(cid:9)2 to 5(cid:9)2 kcal/mol. The Asp and Asn
Anotherlongrangeeffectisthecouplingbetweenthelabile dissociationconstantsthendifferbyafactorofbetween10(cid:10)2.2
His-448protonandthethirdMg2(cid:1)cation.Indeed,wepredict and10(cid:10)5.1.Therangeofvaluesreflectstheuncertaintyinthe
(36)thatwhenATP(cid:2)Mg2(cid:1)(butnotATP(cid:2)Mg2(cid:1))bindswithAsn MDFEestimation.Ifthethirdcationwereabsent10%ofthe
3 2
insteadofAsp,His-448willreverttoitsneutralform.Similarly, time, and we averaged correspondingly over the 2- and
His-254ispredictedtobeneutralwhenthethirdMg2(cid:1)ispres- 3-cation states, the average Asp and Asn dissociation con-
entandpositivewhenitisabsent. stantswoulddifferbyafactorofbetween10(cid:10)3.2and10(cid:10)6.1.
Mg2(cid:1) Binding Is Governed by Charge Balance and Long This is roughly comparable with the average error rate in
RangeInteractions—TheATPphosphategroupsaredeproto- proteinsynthesis,0.03%(cid:4)10(cid:10)3.5.
nated at physiological pH, so that ATP interacts readily with ThisestimateassumesthatHis-448ispresent.Infact,His-
cations. In solution, ATP usually binds one divalent cation. 448isconservedinprokaryotesbutabsentineukaryotes.For-
Whencomplexedtoaprotein,itusuallybindsoneortwo(25). tunately,wefoundthatAspRSis,infact,protectedagainstthe
Yetoursimulationsshowthatthe3-cationformisoverwhelm- 2-cation state, which is negligibly populated in all cases. By
inglyfavoredinAspRS.Giventheroleofthethirdcation,M3,in eliminatingthe2-cationformandalwaysbindingATP(cid:2)Mg2(cid:1),
3
AspRSspecificity,wewanttounderstanditsstability. AspRS ensures a large Asp/Asn discrimination even in
ATPbindspreferentiallytoall10classIIaaRSswitheither eukaryotes,whereHis-448isabsent.
threedivalentcationsortwocationsandapositiveproteinside TheMg2(cid:1)cationsandtheconservedsidechainsthatcoor-
chain(seethedatabaseanalysisinthesupplementalmaterial). dinate them are required for AspRS activity, and the cations
The fully bent, U-shaped geometry of ATP in AspRS is also playbothastructuralandacatalyticrole.Itseemseconomical
characteristicofclassIIaaRSsandmightbethoughttoplaya that the enzyme should also use them for a third purpose, to
roleinM3stabilization.ExcludingaaRSs,onlytwoofthe238 helpensuresubstratespecificity.Incontrast,theevolutionarily
nonredundant NTP(cid:2)protein complexes in the Protein Data related LysRS also binds ATP only in the 3-cation form, but
Bankexhibitsuchafullybentform(seesupplementalmaterial). artificiallyremovingthethirdcationdoesnotaffectbindingof
ThemostobviousM3interactions(Fig.3)arewiththeATP the(positivelycharged)Lyssubstrate.
(cid:2)-and(cid:3)-phosphatesandsurroundingwaters,andATP(cid:2)Mg2(cid:1) Fig.4summarizesthesubstratebindingspecificityinAspRS
2
doesmakeaverylargecontributiontoM3binding((cid:1)367kcal/ asdeterminedfromthepresentsimulationsandearlierstudies.
mol; Table 6). But these groups are not unique to AspRS or Manydifferentstatesarepotentiallyavailable,themostimpor-
aaRSs,sothatthestabilityofM3issomethingofapuzzle.In tantofwhichareshowninFig.4.Someofthemproducealow
fact,longerrangeinteractionsareatplay.Thesewereidenti- Asp/Asnspecificity,andthesestatesareinvariablyunstableand
fiedbythegroupdecompositionofthebindingfreeenergy. negligiblypopulated.Overall,adelicateinterplaybetweencon-
A large contribution ((cid:1)126 kcal/mol) comes from the con- formationalshifts,ATP(cid:2)Mg2(cid:1)binding,andhistidinecharging
3
servedGlu-482andAsp-475sidechains,6and9Åawayfrom allowsAspRStocombineamoderateAspbindingaffinitywith
AUGUST18,2006•VOLUME281•NUMBER33 JOURNALOFBIOLOGICALCHEMISTRY 23801