The Rodin-Ohno hypothesis that two enzyme superfamilies descended from one ancestral gene

23 Pages·2014·3.08 MB·English

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RESEARCH Open Access
The Rodin-Ohno hypothesis that two enzyme
superfamilies descended from one ancestral
gene: an unlikely scenario for the origins of
translation that will not be dismissed
Charles W Carter Jr1*, Li Li1, Violetta Weinreb1, Martha Collier1, Katiria Gonzalez-Rivera1, Mariel Jimenez-Rodriguez1,
Ozgün Erdogan1, Brian Kuhlman1, Xavier Ambroggio2, Tishan Williams1 and S Niranj Chandrasekharan1
Abstract
Background: Because amino acidactivationis rate-limiting for uncatalyzed protein synthesis, it is a key puzzle in
understanding theorigin of the genetic code. Two unrelated classes (Iand II)of contemporary aminoacyl-tRNA
synthetases (aaRS) now translate the code. Observingthat codons for the most highly conserved, Class Icatalytic
peptides, when read in thereverse direction, are very nearly anticodons for ClassII defining catalytic peptides,
Rodinand Ohnoproposed that thetwosuperfamiliesdescendedfrom opposite strands ofthesame ancestralgene.
Thisunusualhypothesis languished for a decade, perhaps because it appearedto be unfalsifiable.
Results: The proposed sense/antisense alignment makes important predictions. Fragments that align inantiparallel
orientations, and containthe respective active sites,should catalyze thesame two reactions catalyzed by
contemporary synthetases. Recent experiments confirmed that prediction. Invariant cores from both classes, called
Urzymes after Ur=primitive, authentic, plus enzyme and representing~20% ofthecontemporary structures, can
be expressed and exhibit high, proportionate rate accelerations for both amino-acid activationand tRNA acylation.
A major fraction (60%)of the catalytic rate acceleration by contemporary synthetases resides in segments that align
sense/antisense. Bioinformatic evidence for sense/antisense ancestryextendsto codons specifyingtheinvariant
secondary and tertiary structures outside the active sites of thetwo synthetase classes. Peptides from a designed,
46-residue gene constrainedby Rosetta to encode Class I and II ATP binding sites with fully complementary
sequences both accelerateamino acid activation by ATP ~400 fold.
Conclusions: Biochemical and bioinformatic results substantially enhance the posterior probability that ancestors
ofthe two synthetase classes arose from opposite strands of thesame ancestral gene. The remarkable acceleration
byshort peptides ofthe rate-limiting step in uncatalyzed protein synthesis,together with thesynergy ofsynthetase
Urzymes and theircognate tRNAs, introduce a new paradigm for theorigin ofprotein catalysts, emphasizethe
potential relevance of anoperationalRNA code embedded inthe tRNA acceptor stems, and challenge the
RNA-World hypothesis.
Reviewers: This article was reviewed byDr. Paul Schimmel(nominatedby Laura Landweber), Dr. Eugene Koonin
and Professor David Ardell.
Keywords: Aminoacyl-tRNA synthetases, Urzymes, Genetic code, Origin ofTranslation, RNA World hypothesis,
Amino acid activation, Structural homology, Ancestral genes, Sense/antisense coding
*Correspondence:[email protected]
1DepartmentofBiochemistryandBiophysics,CB7260UniversityofNorth
CarolinaatChapelHill,ChapelHill,NC27599-7260,USA
Fulllistofauthorinformationisavailableattheendofthearticle
©2014Carteretal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
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reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain
Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,
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Open peer review
Reviewed by Dr. Paul Schimmel (nominated by Laura
Landweber), Dr. Eugene Koonin and Professor David
Ardell. For the full reviews, please go to the Reviewers'
Reportssection.
Dedication
“…thereisnosinglepathtocreativity. Weare
constrained notbythenecessarydisciplineofrigor
butbythelimitsofourown imaginationsand
intellectual courage. Inthe wordsofJazzmusician
FatsWaller,Daretobewrongoryou mayneverbe
Figure1Aminoacyl-tRNAsynthetaseshavethreeimportant
right.”
functionsinthecell.TheyuseATPtoactivatethealphacarboxyl
group,makingtheaminoacidmorereactive(Activation).This
- J.MichaelBishop[1] activationstepaddstheadenosinemoietyasan“affinitytag”tothe
aminoacid,increasingitsbindingaffinityby~1000-fold(Retention)
Thisslowsthereleaseofaveryreactivespeciesandenables
subsequentstepsthatenhancethefidelityofthefinalstep.Finally,
“Howoften haveIsaid toyouthatwhen youhave theycatalyzetransferoftheactivatedcarboxylgrouptothe3’CCA
eliminatedtheimpossible, whateverremains,however terminusoftRNA(Acylation),completingthetranslationofthe
improbable, mustbethetruth?” geneticcodebythecovalentlinkagetothetRNAanticodonthatis
interpretedbythe30Sribosomalsubunitinresponsetocodonsin
mRNA.Theapproximaterateaccelerationsachievedbycontemporary
-SirArthurConanDoyle [2]
enzymesindicatedarebasedoncomparisonsofkcat/K valuestothe
M
uncatalyzedrates,estimatedfromexperimentalratesofmodel
Sergei Rodin (1947-2011) was both a mentor and a reactions,asdescribedin[38,39].
collaborator. When the paper that launched this work
[3] was challenged [4], Sergei was so incensed that he
andhisson,Andrei,wroteabrilliant,rebuttalonourbe- NTP hydrolysis into the highly unfavorable bond forma-
half [5].Thus,Ialsoconsideredhimmyfriend. tion leading to mixed anhydride aminoacyl-5’adenylates,
The origin of the universal genetic code is one of the (ii) addition of a covalent tag to the amino acid, i.e., the
most important, fascinating, and vexing questions facing adenosine moiety, greatly extending the residence time
contemporary biologists. Sergei devoted much of his oftheactivatedaminoacidwithintheactivesitetoallow
professionallifepursuingthisquestion usingseveralper- dissociation and/or hydrolysis of incorrectly activated
spectives from “outside the box” [5-11]. One of his more amino acids [17,18], and (iii) specific acylation of tRNA
unlikely hypotheses was the possible ancestry of Class I with cognate amino acid. Translation occurs in the third
and Class II aminoacyl-tRNA synthetases as sense- and step[19-26].
antisense- gene products expressed from the same prim- Absent catalysts, aminoacyl-5’ adenylates form both
ordial gene [11]. That hypothesis was an elegant very slowly and in very low equilibrium yield. The acti-
realization of thoughts expressed by both Bishop and vation step proceeds 103-104 times more slowly than the
Doyle. This review considers recent experimental sup- second in aqueous solutiona, and therefore requires a
portfor,andimplicationsof,theirhypothesis. correspondingly more potent catalyst. Release and sub-
sequent hydrolysis of the pyrophosphate leaving group
Background are both necessary to ensure that activated amino acids
The origin of life has challenged scientific investigation are formed in high yield. Further, once formed, the
for a century or more, generating diverse opinions relat- aminoacyl-5’ adenylate is exceeded in reactivity only by
ing to several disjoint questions about life’s definition in acyl-halides [27]. In fact, of all reactions involved in
terms of compartmentation, metabolism, and informa- ribosomal protein synthesis and in both kinetic and
tion transfer/storage [12-16]. A central issue linking thermodynamic terms,aminoacidactivation is,mechan-
these questions is a transition from chemistry to biology. istically,byfarthemostchallenging.
Our focus is the origin of codon-dependent translation. Just how far back in time the three functions can be
Contemporary Aminoacyl-tRNA synthetases (aaRS) ac- tracedliesclosetotheheartofthecode’sorigins.Consen-
celerate two successive chemical steps: amino acid acti- susholdsthataaRSenzymeshadessentiallyassumedtheir
vation and tRNA acylation. These coupled tasks involve modernconfigurationsinthelastuniversalcellularances-
(Figure 1):(i) transductionofthe chemicalfree energy of tor, LUCA [28-30]. It is certainly not idle speculation,
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therefore, that simpler ancestral aaRS preceded LUCA by binary codeoftwoaminoacid TYPESthat specify
eons. Nevertheless, because LUCA represents a localized “inside”and “outside”seems torepresentalmost
“Big Bang” [31,32], it was associated with intense genetic sufficientinformation(turnsexcepted)toencode
exchange [33]. Thus, it becomes much more difficult to globularobjectswith selectablefunctionsand hence,
trace phylogenetic lineages for either activity much be- tolaunchnaturalselection. Combinatoriallibraries
yondthathypotheticallandmark.Onepossibleavenuelies ofpolypeptides basedonabinary,middle-base code
in the identification and functional annotation of broadly thatdifferentiates onlybetweencoreand surface
conserved tertiary packing motifs, illustrated pointedly aminoacidscontainhighproportionsofproducts
[34] by a nearly invariant core packing motif belonging with thebiophysical characteristicsofmoltenglob-
to~125 families in the Rossmannoid superfamily. That ules[45],and giverisetosignificantfunctionalities
motif is associated with a discrete supersecondary struc- [46,47].Twodifferentkindsofsynthetaseswithrudi-
turethatbindsATPandnucleotidesingeneral,inkeeping mentaryspecificitiesreflectedinmedian hydropa-
with its possible role in primordial chemical free energy thies ofthe contemporaryClassIand IIaaRS
conversion, and has been identified as a “protoallosteric substratesmight thushavebeensufficient tolaunch
site” [35]. A related effort is the expression and engineer- codon-directedprotein synthesis.
ing of invariant cores from enzyme superfamilies [36-40]. ii. Physicalchemistry.Amino acidsubstrates ofthetwo
We call these constructs Urzymes, from Ur=primitive, classessortintojust suchadistinction.The
original;theyareourcentralfocushere(seealso[41]). apparent symmetryrelatingthe three subclasses and
theinordinatewaterpreference ofClassIarginine
Results [48]maskan overwhelmingdifferencebetweenthe
Aminoacyl-tRNAsynthetases:whytwofamilies? hydrophobiccharacter ofClassIAand ClassIIA
The activation and acylation reactions are so important, aminoacids.Despiteexceptions,ClassIIamino
and the twenty canonical amino acids so diverse, that na- acids generallyprefertheaqueous phase,ClassI
tureinventedtwodifferentaaRSfamiliestoactivateall20 aminoacidsthe hydrocarbonphase.Theirmedian
canonical amino acids. The two enzyme families differ freeenergiesoftransferof betweenwaterand
markedly in primary, secondary and tertiary structure cyclohexanediffer by-4.6 kcal/mole[40].ClassI
[42]. Amino acids activated by Class I and II aaRS divide (larger)andClassII(smaller) amino acidsarealso
intoapparentlysymmetricalclassesof10eachb,withthree distinguished bysize. Solvent transferfreeenergy
comparably sized subclasses, I,IIA(6), I,IIB(2-3), and I,IIC (P<10−7)and massdifference (P<10−4)contribute
(1-2). synergistically(P<10−2)tothe solvent accessible
Class I and II active sites exhibit several relevant surface areainfoldedproteins(Carter,CWJr&
distinctions (Figure 2). First, the amino acid-binding Wolfenden, RtRNA Acceptor-Stemand Anticodon
pocketsofClassIenzymesliedeepwithintheRossmann BasesFormIndependentCodesRelatedtoProtein
dinucleotide-binding fold, whereasamino acids bind to a Folding,inpreparation); ClassIIamino acidside
shallow crevice close tothesurfaceofClassIIenzymes. chainsare,onaverage,54%exposedwhereasClassI
The most highly-conserved aaRS active-site amino aminoacidsare 32%exposed(P~0.03).
acids occur in three sets of signature sequences. We’ll iii.Geneticlinkage.Apre-cellularworldpopulatedby
focus on Class I HIGH and KMSKS and Class II Motif 1 quasispeciesmay haveplacedapremiumonefficient
and 2.Withrare exceptions, conserved amino acids with informationstorage[49].Wefurtherbelievethat a
a direct, catalytic role in Class I active sites are drawn substantialselectiveadvantagewouldarise if both
from amino acid substrates activated by Class II en- requiredkindsofsynthetaseswerepresentatthe
zymes, and conversely (Figure 2). It is hard to imagine same timeand place.Coding ClassIandIIon
how this came about unless the evolutionary ancestors oppositestrandswouldlinkgenesforthe twoclasses
appeared simultaneously, rather than sequentially, as is genetically, assuring thatwhen onewaspresent,so
often argued[43,44]. wasthe other.
In any case, sharply contrasting architectures render it
inconceivable that the two classes had a common ances- Sense/antisenseancestry
tor.Why,then,didnatureinventtwowaystodothesame Rodin and Ohno observed a statistically significant com-
job? Three related aspects of the class division seem rele- plementarity between consensus coding sequences for
vanttoansweringthisquestion: class-I defining PxxxxHIGH and KMSKS peptide signa-
tures and Class II Motif 2 and Motif 1 sequences, and
i. Coding simplicity.Theearliestproteins probably conversely [11]. They inferred from this that ancestral
wereencodedbyamuch simpler geneticcode than Class I and II aaRS descended from opposite strands of
the code of20aminoacids wehavetoday.Infact,a the same gene, a proposal we call the Rodin-Ohno (RO)
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Figure2DetailedcomparisonbetweenactivesitesofClassIandClassIIaminoacyl-tRNAsynthetases.SubstratebindingsitesforATPand
aminoacidareburiedinClassI,andmuchclosertothesurfaceinClassIIenzymes.Thecoloroftheactive-sitespheresillustratesthatconserved
active-siteresiduesthatmotivatedRodinandOhnotoadvancetheirsense/antisensecodinghypothesisaredrawnexclusivelyfromthesetof
substratesactivatedbytheotherclass.ThemediantransferfreeenergyfromwatertocyclohexaneisfavorableforClassIsubstratesandunfavorable
forClassIIsubstrates.
hypothesis. Despite the strength of their statistical tests to perform similar analyses. Contemporary aaRS are mod-
(vide infra), it was not obvious when the idea first ap- erately large enzymes, in keeping with their sophisticated
peared that experiments could either falsify or confirm tasks. Their structures also exhibit considerable variation
thisextraordinaryproposal.Intheinterim, however,spe- withinthetwoclasses(Figure3).Fromtheoutsidethefour
cifying the hypothesis more precisely in terms of the re- ClassIandIImonomerssuperimposedineachpartofthe
spective tertiary structures has clarified its more important figure look quite different. Inside, however, a much
implications, opening experimental and bioinformatic ave- smaller, invariant core of ~120-130 amino acids is nearly
nuestoassessitsvalidity. identicalinall10examplesofeachclass.
Key to these new developments is the notion that con- Not surprisingly, cores for each class encompasses the
temporary enzymes grew from ancestral forms similar to active sites for amino acid activation and acyl-transfer,
invariant cores that can be identified within superim- and hence also the most highly conservedsecondary and
posed members of protein superfamilies (Figure 3). tertiary structures. Urzymology consists of the methods
These cores invariably comprise the active sites. They wehaveintroducedtoidentify,re-design,express,purify,
can be expressed in soluble form, sometimes after re- characterize, authenticate, and exploit the properties of
design to modify hydrophobic side chains at newly these cores (Table1).
solvent-exposed surfaces [39]. They exhibit substantial Whereas invariant cores within each class are very
rate accelerations for the reactions catalyzed by the en- similar to each other, they retain sufficient identity that
zymes from which theyderive [37-40]. sub-class relationships are preserved. Four examples
In the following, we first summarize the salient features (two from subclass A, and one each from subclasses B
of Urzymology, the study of Urzymes. Then we describe and C) were superimposed by partial order structure
howUrzymologyfacilitatesthemodulardeconstructionof alignment(POSA;http://fatcat.burnham.org/POSA;[52])).
Class I TrpRS and Class II HisRS and recapitulation of Urzymes deduced for members of the same subclass (IA,
their evolution. Finally, we summarize how these two IIA) were more similar to each other than to members of
UrzymeshelpvalidatetheRodin-Ohnohypothesis. other subclasses, and the subclassesICand IIC (Figure4)
weremoststructurallydistinct.
Urzymology:structuralbiologyyieldsinsightsaboutthe We examine implications from structural biology for
invariantcores the RO hypothesis in Figure 5. Class I and II invariant
Comparativeanatomyhasalwaysbeenthesinequanonof cores themselves actually can be aligned sense/antisense
phylogenic inference. Because our interest here concerns as noted by Rodin and Ohno [11]. Contemporary aaRS,
molecules far earlier than LUCA, our approach begins however, violate this alignment in two ways. (i) Large and
withstructuralbiologyand3Dsuperposition,whoseappli- variable insertion domains (in Class I these are called
cation to aaRS has been reviewed [50]. We used a variety ConnectingPeptides1and2[53,54]),interrupttheactive-
of manual least squares and automated algorithms [51,52] site alignment. (ii) Anticodon-binding domains in both
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Figure3SuperpositionoffourClassIandfourClassIIaaRS.Specificenzymesarecoloreddifferentlyandlabeled.Full-lengthcontemporary
monomersareshownassurfacesthatare65%transparent,torevealtheinvariantcores,shownascartoonsinsidethesurfaces.
Classes lie outside the range where antiparallel alignment AARSUrzymesbothactivate,andacylatetRNAwith,
isevenpossible. cognateaminoacids.
On the other hand, removing all insertion and Aminoacidactivation
anticodon-bindingdomainsfrombothclassesleavesthe Two Class I (TrpRS and LeuRS) and one Class II
potential sense/antisense alignment intact (Figure 5B). (HisRS) Urzymes accelerate the rate-limiting amino acid
Remarkably, both invariant cores include complete ATP- activation reaction ~108-109-fold. These rates are within
and amino acid-binding sites, together with rudimentary 10−5 (Figure 6; Figure 5 in [37]), so transition-state
binding sites for the 3’ CCA termini of tRNA (Figure 5C; stabilization free energies of both Class IandII Urzymes
[40]). aretherefore ~60%of,those offull-lengthaaRS.
Figure 5C illustrates the chief experimental prediction
ofthe RO hypothesis:parts ofeithergene that cannotbe tRNAaminoacylation
relatedsense/antisense—bothanticodon-bindingdomains, Catalysis of amino acid activation by aaRS Urzymes left
theinsertionsandClassIIMotif3—appearinsomesense a key question unanswered: do these peptide catalysts
functionally superfluous. Removing them leaves precisely also accelerateaminoacylation oftRNA?Acentralimpli-
the invariant cores we had identified for both enzymes, cation of the RO hypothesis is that sense/antisense
and these alignquite closely, sense/antisense.Thekey ex- encoded fragmentsshould beexhibitboth activities.Pre-
perimentalquestion is:how activeare ClassI and IIaaRS cedent and structural arguments led us to expect recog-
Urzymes? Too little catalytic activity to produce activated nition of tRNA by aaRS Urzymes, even without the
aminoacidsata sufficientratetosupport uncatalyzedas- anticodon-binding domain, which is often considered a
sembly into peptides would effectively falsify the RO hy- late addition [23]. A considerable literature describes the
pothesis.Urzymes are catalytically very much more active acylation of isolated tRNA modules containing the ac-
thannecessary. ceptor stem [19,20,55,56]. Comparable experiments have
Table1ProceduresusedinUrzymology
Process Remarks
Superimposehomologs POSAserver:http://fatcat.burnham.org/POSA/
IDsharedinvariantsubset Usually10-30%ofmonomericforms
Re-designexposedsurface Proteindesign:Rosettahttp://www.rosettacommons.org/
Clone&Express MaltoseBindingProtein(MBP)fusions;TEVcleavage
Characterize Rateaccelerations,substratespecificities
Authenticate Singleturnoveractive-sitetitration,Steady-statekinetics,geneticmutationandmanipulation
Evolutionaryrecapitulation Multi-scalemodularde-andreconstruction
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be sure that they do not result from a tiny amount–1 in
100,000–of contaminating wild-type enzyme? Seven dif-
ferent tests (Table 2) argue from multiple points of view
that these activities are authentic. The last four func-
tional assays uniquely distinguish Urzymes from native
aaRS,andarediscussedindetail:
1. Empty vector controls showessentially noactivity.
WeexpressallUrzymesasmaltose-bindingprotein
(MBP)fusions toimprovesolubility. Nounfused
MBPexpressed andpurifiedinthe same manner on
anamyloseresin exceededbackgroundwhenassayed
at12mg/mlwith32PPiexchangemixesfortrypto-
phan,histidine,and leucine.
2. ActiveTrpRSUrzymecan berenaturedfrom
inclusionbodies[40].Itisunlikely thatnative full
Figure4PartialorderstructurealignmentoffourClassIand length enzyme wouldcontaminate inclusion body
ClassIIUrzymes.Despitethestrongsimilaritiesbetweenthefour preparations.
Urzymesfromeachclass,POSAidentifiesappropriatesub-classification.
3. Cleavage ofMBP fusionproteins releasescryptic
activity.MBPfusions inhibitbothTrpRSand HisRS
until now not been performed with modular fragments Urzymes~50-fold [38,39].Crypticactivity released
of aaRS, owing to the greater difficulty of constructing byTobaccoEtchVirusprotease cleavageofpurified
and purifying proteins, compared to RNA. Further, sim- fusion proteinsimplicates boththepurifiedfragment
ultaneous appearance of a fully-developed genetic code, and protease cleavage(seealso 5below).
depending heavily on binding the anticodon loop of 4. Active-sitetitrationsshowsignificant pre-steady state
most tRNAs [56] is difficult to envision. Accordingly, burstsinsingle turnoverassays,amounting to10-
Giegé, Schimmel, and others proposed that an earlier, 90%ofthe totalnumberofmolecules.Active-siteti-
“operational RNA code” in the tRNA acceptor stem was trations measuresingleturnovertime courses.If
aforerunnerofthe present daycode[23]. productreleaseisrate-limiting,thenturnover will
The crystal structure of human cytosolic TrpRS com- beslowerthanthe firstroundofcatalysis,andex-
plexed with the acceptor stem of tRNATrp [57] affords a trapolationofthesteady-staterate totheorigin can
model for the corresponding interaction with theTrpRS beused toestimate the“burst”orthe amplitudeof
Urzyme (from H. Hu’s MD simulations; [39]) Figure 7A. thefirst-orderportion ofthereaction.Burst size
Buried surface area calculation with a probe radius of thereforeestimatestheproportionofactive mole-
1.5 Å shows that 522 Å2 of the Urzyme is potentially in cules.Contaminatingactivity from atiny amount of
contact with tRNATrp. Further, complementary tRNA- wild typefull-length enzyme 105-fold moreactive
binding surface in the α-helix of the second crossover thanthe Urzymeswouldexhibitaninsignificant
connection of the Urzyme includes an invariant, E152, burst,andthe entiretime course wouldrepresentits
that specifically recognizes the discriminator base A73. steady-state rate.Activefractionsalsoprovide more
Similar considerations apply to the HisRS Urzyme(cid:129)tR- accuratek values.
cat
NAHiscomplex(Figure 7B). BothUrzymesshowsubstantialbursts,whichrange
Data shown in Figure 7A, 7B for 32P-3’ adenosine- between~10and90%—much biggerthanthose
labeled tRNATrp and tRNAHis [58] demonstrate that expectedfromarare,veryactive contaminant.Full
TrpRS [39] and HisRS [38] Urzymes catalyze tRNA ami- length aaRSbindtightlytothe aminoacyl-adenylate
noacylation. TrpRS and HisRS Urzymes therefore retain toprotectthe cellfrom ahighlyreactiveadenylating
afullfunctionalrepertoire.Inparticular,catalyticactivities reagent andto preservethespecificity achievedby
required for protein synthesis are as finely tuned as the theactivationstep [17].Itisremarkablethat the
contemporary enzymes, between activation and acylation Urzymesalsosequester theintermediate. Pre-steady
and between Classes, [37]. They therefore represent con- stateburstsarethus athirdkey function offull-
vincingmodelsforancestralClassIandClassIIaaRS. length aaRS(Figure1)retained byUrzymesfrom
bothClasses.
AreUrzymeactivitiesauthentic? 5. Active-sitemutantsandmodular variantshave
Urzyme catalysis of amino acid activation is ~105-fold altered activities.Molecular biologistsrecognizethat
weakerthanthatofcontemporaryenzymes. Howcanwe manipulating thegeneofa suspectedsourceof
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Figure5TheRodin-OhnohypothesisholdsthatancestralformsofClassIandIIaminoacyl-tRNAsynthetases(aaRS)hadfully
complementarynucleicacidcodingsequencesandthatcontemporaryaaRSdescendedfromoppositestrandsofasinglegene.The
schematicinAillustrateshowthishypothesisleadsdirectlytotheconceptofaaRSUrzymes.Antiparallelalignmentofaminoacidsequencesfor
theClass-definingmotifs(HIGHandKMSKSfromClassI;Motifs1and2fromClassII)revealsthatneitheranticodon-bindingdomain,noralong
insertionineachClasscanphysicallybeapartofsuchanancestralgene.Asaresult,theonlyportionsofthetwoClassesconsistentwiththe
hypothesis(B)areabout120-130residueslong.Thesefragmentscoincidewithinvarianttertiarystructuralcoressharedbyallsuperfamilymembers.
Moreover,thesesegmentstogethercomposeaminimalactivesite,containingbindingsitesforallthreesubstrates(C).AminoacidandATP
determinantsarereflectedacrossthegenesequence,whiletRNAbindingdeterminantsarerelatedbytwo-foldrotation[40].
activity canimplicatethatgene product inthe significantamount(Figure8;[38]).WePCR
observedactivity.Wethereforetestedactive-site amplifiedthe122residue fragmentcontainingonly
mutationsand modular variantsintheTrpRSand Motifs 1and 2,addingeithera six-residueN-
HisRSUrzymes.Allsuchexperimentssignificantly terminalextension(red),orMotif 3(yellow),or
alteredactivity. Oneresult—mutationD146Ain both,giving usabalanced assayforthe effectsof
TrpRSUrzymeactuallyincreased, rather thanredu- bothfactors.Theintrinsiccatalyticenhancementof
cing activity asitdoesintheWT enzyme—was Motif 3totransition-statestabilizationbyHisRS
counter-intuitive.However,the catalyticfunction of Urzyme,-0.85 kcal/Mole,isessentiallyidenticalto
D146 infull-lengthTrpRSlikelyrequiresallosteric thatofthemuch shorterandlessobviousN-
couplingofmissingCP1 and ABDmodules,and its terminalextension;their synergisticeffectisnearly
presenceintheUrzyme likelystabilizes theground twicethat.Figure 8emphasizes thatthetwomod-
state, ratherthanthe transitionstate [59]. ulesstabilize theATP bindingsitefrom opposite
Ofparticularinterest arethermodynamiccyclesthat faces ofthe molecule.
involve anaaRSUrzymeand complementing FullTrpRSspecificityand tRNATrpaminoacylation
segments.Veryshort(6-20aa) modules accelerate activity both require essentially complete
HisRSUrzymeaminoacid activationbya smallbut interdomainsynergy(Figure9;[36]). TheTrpRS
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Figure6QuantitativeframeworkinwhichtoassessthecatalyticsignificanceofUrzymesandvariousotherputativestagesofaaRS
evolution.A.Rateaccelerationsestimatedfromexperimentaldataforsinglesubstrate(red)andbi-substrate(Black,Bold)reactionsadaptedfrom
[75]toincludeuncatalyzedandcatalyzedratesofbi-substratereactionsoftheribosome[74],aminoacidactivation[39]andkinases[106].Second
orderrateconstants(blackbars)wereconvertedintocomparableunitsbymultiplyingby0.002M,whichistheATPconcentrationusedtoassay
thecatalystsshowninB.B.Experimentalrateaccelerationsestimatedfromsteadystatekineticsaskcat/K foraseriesofcatalystsderivedfrom
M
ClassIandClassIIaminoacyl-tRNAsynthetases([38,39]anddataofV.Weinreb,L.Li,M.Collier,andK.Gonzalez-Riverapresentedhereinasubsequent
section).VerticalscalesinAandBarethesame,andtheoriginofthehistograminBhasbeensetequaltotheuncatalyzedrateofaminoacid
activationin(AAact)inA.RedbarsdenoteClassITryptophanyl-andLeucyl-tRNAsynthetaseconstructs,bluebarsdenoteClassIIHistidyl-tRNA
synthetaseconstructs,andgreendenotesaribozymalcatalyst[97],includedforcomparison.ResearchpresentedinA,Bwasoriginallypublishedin
[37].©TheAmericanSocietyforBiochemistryandMolecularBiology.C.ClassILeuRSandClassIIHisRSUrzymeaminoacidspecificities.Aminoacids
withmorenegativeΔGk /K valuesindicatehigheractivities.By~1kcal/mole(lightbands)or~five-fold,eachUrzymepreferssubstratesfromthe
cat M
classtowhichitbelongs(darkbands).Nonetheless,bothactivatearangeofnon-cognateaminoacids,andarepromiscuous.
Urzyme favors tryptophanactivationby~10-fold peptide segmentsthatcanalignantisense tothe
overcompeting tyrosine and is~400timesless HisRSUrzyme, isactuallybetter atthe twotasks—
specificthanfull-lengthTrpRS.CP1 andthe amino acidrecognition and tRNA aminoacylation—
anticodon-bindingdomain (ABD) mustaccount for requiredofaaRS,and hencelie closer totheactual
theincreasedspecificity ofnativeTrpRS.Wead- pathofaaRSevolutionthaneitherintermediate,
dressed thisquestionbycomparing specificitiesof potentiallymoreadvancedconstruct.Evolutionary
intermediate constructsinwhich theCP1and ABD developmentofcontemporaryenzymes mustbe
modules wereadded backindividually tothe moresubtlethansimplyaccumulating onemodule
Urzyme [36]toformacompletefactorialexperi- atatime.
ment inthosetwovariables(Figure9A). 6. Steady stateK valuesdiffer fromthose oftheWT
M
Quitesurprisingly(Figure9B),although addingback enzymes.Enzymologistsrecognize thatthesteady-
eitherCP1 ortheABD doesenhance tryptophan stateK valueisan independent signature.
M
binding,thiseffectisnon-specific.Additionofeither Contaminatingwild-type enzymes,irrespectiveof
domain alsoreduced K fortyrosine,suchthat the concentration, wouldsaturateatthesame amino
M
logofthespecificity ratio(k /K ) /(k /K ) , acid concentrations.AlteredK values arethus
cat M Trp cat M Tyr M
wasactually~0.0 (Figure9B).The ~400-foldin- strong evidenceagainstcontaminating wild-type en-
creaseinspecificityobservedforfull-lengthTrpRS, zyme activity. @ATP-and amino acid-dependent
relative totheUrzymedependsentirely oncoopera- Michaelis-MentendatafortheTrpRSand HisRS
tiveinteractions(alsocalled epistasis[60-62])be- Urzymesshow thatATPbindingaffinity iseitherthe
tween thetwodomains[36]. same ortighter toUrzymesthan tocontemporary
tRNATrpaminoacylationrequirescomparable aaRS[38,39].Thek values arenearlycomparable
cat
interdomainsynergy(Figure9B).These experiments tothose ofthenative enzymes. Urzymeaminoacid
withTrpRSUrzymesupporttheunexpected K s,however,are quitedifferent fromthose offull-
M
conclusionthattheUrzymeitself,consistingonlyof length,nativeenzymes.TheTrpRSUrzymeK for
M
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Figure7tRNATrpAcylationbyTrpRSUrzyme.A.ModeloftheputativeinteractionbetweenTrpRSUrzymeandtRNATrp,derivedfromthe
crystalstructureofthecomplexbetweenhumanTrpRSandtRNATrp[57].AutoradiogramsdocumentingtheacylationoftRNATrpbyWildType
TrpRS,TrpRSUrzyme,andtwointermediatemodularconstructs,containingeitherCP1ortheanticodon-bindingdomain.B.Modelfor
interactionoftRNAHiswithHisRS4UrzymeandautoradiogramsshowingacylationbyHisRS1,2,and4asinA.SpheresshowTrp-5’AMP,and
His-5’sulfoamyladenylate.Thesedatawerepublishedoriginallyin[37]©TheAmericanSocietyforBiochemistryandMolecularBiology.
Table2CriteriafortheauthenticityofUrzymecatalyticactivities
Criterion Implementation Remarks
Emptyvectorcontrols Purify,assayMBP DeRigueur,butunconvincing
Renaturationfrominclusionbodies TaggedUrzymespurifiedfrom WTEnzymesdonotsegregatewithinclusionbodies
pellet
MBPfusionsreleasecrypticactivityonTEV Assayfusionproteins±TEV Inhibitioninfusionproteinsiswidespread,notuniversal.
cleavage. cleavage
Active-sitetitrationsUrzyme Singleturnovertime-courses Akeycriterion,thisisalsoessentialforcomparingk /K .
cat M
preparationshavesignificantbursts.
Mutations,modularalterationsinduce Determineeffectofactive-site Active-sitemutationsgenerallyaffectUrzymeactivitiesdifferently
predictablechangesinactivity. mutations,geneticmanipulations andcanactuallyenhanceactivitybecausemechanismsaredifferent.
Urzymes,WTenzymeshavedifferent Measure:k ,K ,k /K ContaminationbyWTenzymewouldsaturateatWTK .
cat M cat M M
Steady-stateK values.
M
Aminoacidspecificityisdifferent Compare:(k /K ) /(k /K ) Urzymesaregenerallylowspecificity,highk catalysts.
cat MW cat MY cat
fromfull-length
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tryptophanis ~1mM,500 timeshigherthan thatof
wild typeTrpRS[39].That forHisRS-3,containing
Motifs 1,2,and3,butlackingthesix-aminoacid N-
terminalextensiontoMotif 1,is120μM, compared
to30 μMforwild-typeHisRSand45 μMforthe
N catalyticdomain [38].
cat
7. HisRSandTrpRSUrzymeshave reduced,but Class-
dependent specificities.Weaker aminoacid affinities
imply thattheUrzymeslikelyhavereduced
specificity fortheircognateaminoacids. TheTrpRS
Urzymeretainsa10-fold preferencefortryptophan
vstyrosine[36,39].Second-order rate constant free
energies,-RTln(k /K ),foraminoacid substrates
cat M
activatedbyClassILeuRSand ClassIIHisRS
Urzymes(Figure6C)showthatbothUrzymesare
promiscuous.However,they bothpreferentiallyacti-
vate aminoacids similar totheoriginalsubstrate
(i.e., LeuandHis). Byafactor of~5-fold,they both
Figure8Quantitativeanalysisofthecatalyticcontributionsof
preferaminoacid substratesfromtheClasstowhich
Motif3andashortN-terminalextensionoftheMotif1helixto
theaccelerationofhistidineactivationbyHisRSUrzyme. they belong.Thismodest,class-dependent substrate
GraphicsincludetheHistidyl-5‘adenylateasspheres.Detailsofthe specificity alsorulesoutadventitiousactivitiesunre-
constructionsaredescribedin[38]. lated toaaRS-derivedconstructs.Aswithactive-site
titrationand steady-statekinetic parameters,con-
taminatingwild-typeaaRSwouldhavea specificity
of~4000-fold.
Figure9FactorialanalysisofTrpRSinter-domaineffects.A.ThecatalyticactivityoftheUrzymefacilitatesafull-factorialanalysisofthebenefits
ofaddingeithertheCP1oranticodon-bindingdomain,jointlywiththeirsynergisticeffectinthefull-lengthenzyme.B.Freeenergyhistogramsforthe
factorialdesigninA,showingnearlyidenticalpatternsinwhichtheCP1andABDactuallydiminishbothspecificityfortryptophanvstyrosineand
acylationoftRNATrp.TheentiredifferencebetweenUrzymeandfull-lengthenzymeisachievedonlyviathesynergisticparticipationofbothdomains
missingintheUrzyme.