Article Mutational Studies on Resurrected Ancestral Proteins Reveal Conservation of Site-Specific

16 Pages·2015·1.47 MB·English

Mutational Studies on Resurrected Ancestral Proteins Reveal
Conservation of Site-Specific Amino Acid Preferences
throughout Evolutionary History
Valeria A. Risso,y,1 Fadia Manssour-Triedo,y,1 Asuncio(cid:2)n Delgado-Delgado,1 Rocio Arco,1
Alicia Barroso-delJesus,2 Alvaro Ingles-Prieto,1,z Raquel Godoy-Ruiz,3 Jose A. Gavira,4 Eric A. Gaucher,5
Beatriz Ibarra-Molero,1 and Jose M. Sanchez-Ruiz*,1
1DepartamentodeQuimicaFisica,FacultaddeCiencias,UniversidaddeGranada,Granada,Spain
2UnidaddeGeno(cid:2)mica,InstitutodeParasitolog(cid:2)ıayBiomedicinaLo(cid:2)pezNeyraCSIC,PTSGranada,Granada,Spain
3DepartmentofBiochemistryandMolecularBiology,UniversityofMarylandSchoolofMedicine,Baltimore,MD
4LaboratoriodeEstudiosCristalogra(cid:2)ficos,InstitutoAndaluzdeCienciasdelaTierra,ConsejoSuperiordeInvestigaciones
Cient(cid:2)ıficas–UniversidaddeGranada,Granada,Spain
5SchoolofBiology,SchoolofChemistryandBiochemistry,ParkerH.PetitInstituteforBioengineeringandBiosciences,Georgia
InstituteofTechnology
D
yTheseauthorscontributedequallytothiswork. o
w
zPresentaddress:ISTAustria,AmCampus1,Klosterneuburg,Austria nlo
a
d
*Correspondingauthor:E-mail:[email protected]. ed
Associateeditor:AndrewRoger fro
m
h
ttp
Abstract ://m
b
e
Localproteininteractions(“molecularcontext”effects)dictateaminoacidreplacementsandcanbedescribedintermsof .o
x
site-specific,energeticpreferencesforanydifferentaminoacid.Ithasbeenrecentlydebatedwhetherthesepreferences fo
rd
remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such jo
u
research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and rn
a
molecularevolutionmodeling.Here,wetakeadvantageoftherecentavailabilityofphenotypicallysupportedlaboratory ls.o
resurrectionsofPrecambrianthioredoxinsandb-lactamasestoexperimentallyaddressthechangeofsite-specificamino arg/
acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary t G
e
adjustmenttoanewaminoacidmayoccur,buttoalargeextentthisisinsufficienttoerasetheprimitivepreferencefor o
rg
amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evo- ia
In
lutionaryhistorydespitelocalsequencedivergence.Weshowsuchpreferenceconservationtobereadilyunderstandable s
in molecular terms and we provide crystallographic evidence foran intriguing structural-switch mechanism: Energetic titute
A preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the of T
r ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation nat- ec
t urallyleadstooneplausibleevolutionaryexplanationfortheexistenceofintragenicglobalsuppressormutations. hn
i o
c lo
l Keywords:molecularevolution,ancestralproteins,aminoacidreplacements. gy
e o
n
M
a
Introduction rc
interest in understanding the structural, functional, and en- h 1
1
Molecular evolution can be described in terms of modifica- ergeticfactorsthatdeterminethebasicevolutionarymovesin , 2
0
tions in protein (or nucleic acid) sequences that result in protein sequence space, that is, the set of acceptable single 1
6
changesinrelevantmolecularpropertiesthatmayultimately mutations.
impactorganismalfitness(Bershteinetal.2006;Nowak2006; Molecular context is required to understand site-specific
SikosekandChan2014).Evolutionarysequencemodifications aminoacidreplacements.Forinstance,anaminoacidismore
are,inmostcases,singlemutations.Forevolutionbynatural likely to occur in a given position if it generates stabilizing
selectiontooccur,acertainnumberofthesesinglemutations interactions or positive contributions. Such context-related
(at least one) must be accepted by a functional protein. effects are widely recognized in the literature and described
Expressedintermsof theMaynard–Smith’ssequencespace usingavarietyterms:Propensities,preferences,formingten-
concept(Smith1970),functionalproteinsmustformcontin- dencies,andsoon.Weshallusetheterm“preference”here.
uous networks in sequence space withnodes connectedby Amino acid preferences can be estimated from statistical
acceptable single-mutation steps. There is considerable analyses of amino acid occurrence (“statistical” preferences;
(cid:2)TheAuthor2014.PublishedbyOxfordUniversityPressonbehalfoftheSocietyforMolecularBiologyandEvolution.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense
(http://creativecommons.org/licenses/by-nc/4.0/),whichpermitsnon-commercialre-use,distribution,andreproductioninany Open Access
medium,providedtheoriginalworkisproperlycited.Forcommercialre-use,[email protected]
440 Mol.Biol.Evol.32(2):440–455 doi:10.1093/molbev/msu312 AdvanceAccesspublicationNovember12,2014
MBE
MutationalStudiesonResurrectedAncestralProteins . doi:10.1093/molbev/msu312
Chou and Fasman 1974; Richardson JS and Richardson DC are conserved or change substantially during evolution. We
1988) and also from experimental stability measurements, mayexpectenergeticpreferencesatagivensiteinaprotein
suchasmutationaleffectsonstability(“thermodynamic” or tobedeterminedbytheinteractionsofaminoacidsatthat
“energetic”preferences;KimandBerg1993;Smithetal.1994; positionwiththeirneighboringsites(andalsowiththoseat
Myersetal.1997).Goodcorrelationsbetweenstatisticaland distant sites if electrostatic charge–charge interactions are
energetic preferences for amino acids in different types of relevant). Since residues at interacting sites change during
secondary structures have been reported (Pace and Scholtz evolution, it is conceivable that preferences at each given
1988;KimandBerg1993;Smithetal.1994). position alsochange.Indeed,recentcomputationalanalyses
Preferencesforaminoacidsinsecondarystructurescanbe (Pollock et al. 2012) support the notion that preferences
viewed as average or typical values that describe some rele- change after mutation in the direction of making the new
vantgeneraltrends.Morefully,fromarigorouspointofview, aminoacidmoreacceptableovertime,anadjustmentthatis
anyparticularsiteinanyproteinhasanassociatedpreference referredtoasthe“evolutionaryStokesshift.”However,Fersht
scalethatmaydifferfromthepreferencescalesatothersites and coworkers (Serrano et al. 1993) found that effects on
inthesameproteinandalsofromthescalesatcorresponding stability of mutations separating barnase and binase (85%
sites in other homologs. Site-specific energetic preferences sequenceidentity)wereindependentandadditive.Morere-
D
canbeassessedbydeterminingthestabilityeffectsofmuta- cently,experimentalstabilitystudies(Ashenbergetal.2013) o
w
tionsatthesiteandmayshowahighlevelofdiscrimination supported evolutionary conservation of amino acid prefer- n
lo
betweenthedifferentaminoacids.Forinstance,ifpackingata encesforsixmutationsinnucleoproteinsfromfourdifferent ad
e
d
buriedsiteisoptimizedforagiven hydrophobicresidue,re- strains of influenza A virus by claiming to disfavor strong fro
placementwithanother hydrophobic aminoacidof similar, changes in amino acid preferences during evolution. Yet, m
h
butsmaller,sizeislikelytobedestabilizingduetolessefficient this general implication of the mutational data for different ttp
packing(Godoy-Ruizetal.2005).Thispresumablysmalldif- nucleoproteins has been called into question (Pollock and ://m
ferenceinenergeticpreferencemayhaveconsequencessince Goldstein2014).Itisthereforeunresolvedwhetherthepref- be
.o
the stabilities of natural proteins are marginal, that is, only erences for different amino acids at each site in a protein x
fo
slightlyabovetheevolutionarystabilitythresholdforpurifying change during evolution or remain essentially constant. rd
jo
selection(TavernaandGoldstein2002;Bershteinetal.2006; Needlesstosay,thisisacrucialissuethatbearsonthemeth- u
rn
Godoy-Ruizetal.2006;Bloometal.2007;Tokurikietal.2007; odologiesusedforphylogeneticanalysisandthedescription als
.o
SikosekandChan2014),andevenamoderatelydestabilizing and modeling of molecular evolutionary processes. For in- rg
mutationcouldbringstabilitybelowthethresholdandthus stance, if preferences are conserved to a substantial extent, a/
t G
preventproperproteinfoldingorfacilitateproteindegrada- modelsthatassumeindependentevolutionatdifferentpro- e
o
tionwiththeoverallresultthatorganismalfitnesswouldbe tein sites are reasonable. On the other hand, if site-specific rg
ia
compromised and the mutation would be rejected—in the preferenceschangestrongly,widespreadaminoacidcoevolu- In
s
absenceofdrift. tion must be explicitly included in molecular evolution titu
Theabovereasoningdoesnotimplythattheaminoacid models.Atamorefundamentallevel,therecentcontroversy te o
presentatanyparticularsiteisnecessarilytheresidueatthe ontheevolutionaryratesofpreferencechange(Pollocketal. f T
e
c
top of the energetic preference ranking. In fact, several sce- 2012; Ashenberg et al. 2013; Pollock and Goldstein 2014) h
n
o
nariosmayexplaintheacceptanceandfixationofalessen- highlightsourlimitedunderstandingofoneofthefundamen- lo
g
ergetically preferred residue at a given site. For instance, a tal steps in evolution: The replacement of amino acids in y o
n
previouslystabilizingmutationinanotherregionofthepro- proteins. M
teinstructure(aso-calledcompensatingorpermissivemuta- Theavailabilityoflargenumbersofproteinsequences,to- arc
h
tion;Weinreichetal.2006;Bloom etal.2007;Ortlundetal. getherwithadvancesinbioinformaticsandmolecularbiology 1
1
2007;Wyganowskietal.2013)couldenhanceproteinstability methodologies,allowimportantissuesinmolecularevolution , 2
0
in such a way that the mutation to the less energetically to be experimentally addressed on the basis of laboratory 16
preferredaminoaciddoesnotviolatethestabilitythreshold resurrectionsofancestralproteins(PaulingandZuckerkandl
forpurifyingselection.Subsequently,thelesspreferredamino 1963;Benneretal.2007).Recentexamplesincludetheadap-
acidcouldpersistatthesiteifitspresencebringsaboutsome tationofproteinstochangingplanetaryconditions(Gaucher
functional changes that translate into enhanced organismal et al. 2008; Perez-Jimenez et al. 2011; Akanuma et al. 2013;
fitness.Note,however,that,eveninthiscase,energeticpref- Risso et al. 2013; Risso, Gavira, Sanchez-Ruiz 2014; Risso,
erencesplayafundamentalrole,astheydeterminetheevo- Gavira, Gaucher, et al. 2014), the origin and evolution of
lutionary trajectory leading to the acceptance of the less thermophily(Hobbsetal.2012),theoriginofcomplexityin
preferredaminoacid. biomolecularmachines(Finniganetal.2012),theroleofepis-
Overall, there can be little doubt about the relevance of tasis in the emergence of new protein functions (Ortlund
site-specific energetic preferences and, indeed, site-specific et al. 2007), the mechanisms of evolutionary innovation
effectshavebeenincludedinmanyeffortstomodelmolec- through gene duplication (Voordeckers et al. 2012), the
ularevolutionaryprocesses(HalpernandBruno1998;Lartillot degree of conservation of protein structure over planetary
andPhilippe2004;Leetal.2008;Wangetal.2008;Rodrigue timescales (Ingles-Prieto et al. 2013), the evolutionary origin
et al. 2010; Tamuri et al. 2012; Bloom 2014). On the other of detoxifying enzymes (Bar-Rogovsky et al. 2013), and the
hand,itisnotatallclearwhetherthepreferencesthemselves characterization of the evolutionary events leading to gene
441
MBE
Rissoetal. . doi:10.1093/molbev/msu312
silencing(Kratzeretal.2014).Here,wetakeadvantageofthe Kumar 2009). Bayesian ancestral sequence reconstruction
recentavailabilityofphenotypicallysupportedlaboratoryres- was used to obtain probabilistic estimates of the sequences
urrectionsofPrecambrianproteinstoexperimentallyaddress atallthenodesofthephylogenetictrees.Thegenesencoded
theevolutionofaminoacidpreferencesonatimescaleof~4 by the reconstructed sequences at the targeted nodes were
billion years (i.e., the time span of life on Earth). We used synthesized and then expressed by Escherichia coli in the
nearly 200 diverse extant thioredoxin sequences comprising laboratory(ortousetheparlance,“resurrected”)andexhaus-
the three domains of life to construct a highly articulated tively characterized in terms of structure, function, and sta-
phylogenetic tree (Perez-Jimenez et al. 2011). In addition, bility(Perez-Jimenezetal.2011;Ingles-Prietoetal.2013;Risso
weuseda setof 75chromosomalsequences of extantclass et al. 2013; Risso, Gavira, Sanchez-Ruiz 2014; Risso, Gavira,
Ab-lactamasestoconstructaphylogenetictreeencompass- Gaucher,etal.2014).Theywerefoundtoadoptthecanonical
ing Gram-positive and Gram-negative bacteria (Risso et al. foldoftheirmoderncounterpartsdespitealargenumberof
2013). In both cases, the trees were sufficiently close to ac- mutationaldifferences(closeto50%ofthesequenceinsome
cases)andtheirpropertiesledtoplausibleevolutionarynar-
cepted organismal phylogenies to allow us to target
ratives that supported that proteins encoded by the recon-
well-defined Precambrian phylogenetic nodes (fig. 1 ) for
structedthioredoxinandb-lactamasesequencesarecredible
which reliable age estimates are available (Hedges and
D
phenotypic representations of the proteins that existed o
w
billionsofyearsago. n
lo
Theavailabilityofphenotypicallysupportedlaboratoryres- ad
e
d
urrections of Precambrian proteins allows us to address the fro
evolution of amino acid preferences in a straightforward m
h
mannerbecause:(1)Ancestralsequencereconstructionanal- ttp
yses lead to plausible estimates of the “age” of each given ://m
aminoacidinamodernprotein(i.e.,thefirstappearanceof be
.o
theaminoacidalongthelineofdescentfromtheancestorto x
fo
theextantproteinunderstudy)and,therefore,toestimates rd
jo
ofthegeologictimeavailableforenergeticadjustmentand(2) u
rn
measurements of mutational effects on the stability of als
.o
modern proteins can be compared with experimentally rg
determined effects of the same mutations on the stability a/
t G
of the credible representations of their ancestors. Molecular e
o
clock age estimates are available for many Precambrian and rg
ia
Cambriannodesforthetreeoflife(HedgesandKumar2009). In
s
We usetheseestimatesasproxiestobetterunderstandthe titu
geologic timescales associated with site-specific amino acid te o
preference. Given the controversial nature of molecular f T
e
c
clocks, however, we also present our results in the context h
n
o
ofsequencedivergence(fig.1). lo
g
Wefirstreportacomparativeexperimentalanalysisonthe y o
n
effect of 21 mutations on the stability of both E. coli thior- M
edoxinandonthelaboratoryresurrectioncorrespondingto arc
h
the thioredoxin of the last bacterial common ancestor 1
1
(LBCA). The time span of this comparison is billions of , 2
0
years and all the mutations selected involve highly similar 16
amino acids and very minor structural alterations. If our re-
sults demonstrate that amino acid preference is conserved
acrosslongevolutionarytimescales,itmaybereasonableto
inferthatthisisageneralphenomenonthatholdsforshorter
FIG.1. Aminoaciddifferencesbetweenextantsequencesandthere- timescalesand,moreimportantly,fordissimilaraminoacids.
constructedsequencesforthePrecambriannodestargetedinthiswork. Despite the plausibility of this inference, we deemedit con-
AgeestimatesarederivedfromtheTimetreeofLife(HedgesandKumar venient to specifically test preference conservation in in-
2009).(A)Thioredoxins.Theancestralnodestargetedarethefollowing: stances involving the exchange between highly dissimilar
LGPCA, last common ancestor of (cid:2)-proteobacteria; LPBCA, last
amino acids. We thus report the effect of the lysine/leucine
common ancestor of the cyanobacterial, deinococcus, and thermus
exchangeatposition90onthestabilityofE.colithioredoxin
groups. Also shown is Escherichia coli thioredoxin. (B) b-lactamases.
andseverallaboratory-resurrectedPrecambrianthioredoxins,
The ancestral nodes targeted are the following: PNCA, last common
sothattheevolutionaryhistoryoftheKversusLpreference
ancestorofvariousGram-positiveandGram-negativebacteria;GNCA,
canbefollowedacrosslargegeologictimescales.Interestingly,
lastcommonancestorofvariousGram-negativebacteria;GPBCA,last
commonancestorof(cid:2)-proteobacteria;ENCA,lastcommonancestorof the preference conservation found in this case (L is always
enterobacteria.TEM-1referstoE.coliTEM-1b-lactamase. energetically preferred over K at position 90, even for
442
MBE
MutationalStudiesonResurrectedAncestralProteins . doi:10.1093/molbev/msu312
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
://m
b
e
.o
x
fo
rd
jo
u
rn
a
ls
.o
rg
a/
t G
e
o
rg
ia
In
s
titu
te
FIG.2. Ancestralandextantthioredoxins.(A)Schematicphylogenetictreeshowingthioredoxinnodesrelevanttothisstudy(seefig.1fordefinitions). of T
Numbersalongsidethenodesstandforthevaluesofthesequenceidentitywiththeextantprotein.(B)StructuresofE.colithioredoxinandLBCA e
c
h
thioredoxin(PDBcodes2H6Xand4BA7).Positionstargetedformutationareshown(E.colithioredoxinnumbering)withacolorthatreferstothe n
o
relationbetweenthemutationsactuallyperformedinthetwobackgrounds.Blue,samedirectioninbothbackgrounds;red,oppositedirections(see lo
g
maintextandsupplementarytableS1,SupplementaryMaterialonline,fordetails).(C)Fractionofaminoacidspresentintheneighborhood(6A˚)of y o
n
eachLBCAthioredoxinresiduethatareidenticalwiththoseintheneighborhoodofthesamepositioninE.colithioredoxin.Positionstargetedfor M
mutationareshownwithblackbars.Thehorizontaldashedlinecorrespondstothesequenceidentity(0.55)betweenthetwoproteins. arc
h
1
1
, 2
0
1
6
thioredoxinsinwhichthereisaKatthatposition)islinkedto Results and Discussion
an unanticipated mechanism involving a local structural
Comparative Analysis of the Effect of 21 Chemically
switchuponmutation.Finally,weconsidertheeffectofthe
Conservative Mutations on the Stability of the
methionine/threonineexchangeonthestabilityoftwomod-
ernb-lactamases(E.coliTEM-1andBacilluslicheniformis)and Thioredoxins from E. coli and the LBCA
three laboratory-resurrected Precambrian b-lactamases Wepreviouslyreportedtheeffectsofalargenumberofmu-
dating up to about 3 billion years ago. This is a particularly tationsonthestabilityoftheextantthioredoxinfromE.coli
interestingcasebecausetheM182TmutationintheTEM-1 (Godoy-Ruizetal.2004,2005,2006).Allthemutationsstud-
b-lactamase gene is a global suppressor (Huang and Palzkill ied belong to the E!D, D!E, I!V, and V!I types and
1997;Wangetal.2002;Bloometal.2005;Salverdaetal.2010) introduce,therefore,verysmallmolecularchanges:Thepres-
that appears linked to many clinical cases of emergence of enceorabsenceofa–CH inthecaseofanI$Vreplacement
3
resistancetowardnewantibiotics.Infact,ourresultssuggest versusthepresenceorabsenceofa–CH -(andlikelyasmall
2
thatarelationshipbetweenconservationofaminoacidpref- differenceinthespatialpositionofthenegativecharge)inthe
erence and the existence of global suppressor mutations caseofanE$Dreplacement.Here,wedeterminetheeffects
existsforthisproteinfamily. of these mutations on the stability of the laboratory
443
MBE
Rissoetal. . doi:10.1093/molbev/msu312
resurrection of the thioredoxin in the LBCA (fig. 2A).
The mutations performed on the ancestral protein back-
groundcanbeclassifiedintotwogroups.Fourteenmutations
areidentical,intermsoftheresiduesinvolvedandthedirec-
tionofthemutation,withthosewepreviouslyintroducedin
theE.colibackground;forinstance,thereisavalineatposition
16 in both E. coli and LBCA thioredoxins and therefore the
effect of the V16I mutation can be studied in both back-
grounds.Ontheotherhand,sevenmutationsmustbestud-
ied in opposite directions for the extant and ancestral
backgrounds. For instance, there is an isoleucine at position
23 in E. coli thioredoxin, while a valine is present at the
same position in LBCA thioredoxin. The I23V mutation is
performedontheextantbackgroundandV23Iisperformed
on the ancestral background. For comparison purposes, the
D
stability effect of the V23I mutation is changed in sign to o
w
obtain the corresponding value in the “E. coli direction” n
lo
(i.e., I23V). The positions corresponding to these two kinds ad
e
d
ofmutationsarerespectivelylabeledinblueandredinfigure fro
2B (the same color code is used in supplementary table S1, m
h
Supplementary Material online, where the mutations are ttp
described in detail). It must be noted that, as expected ://m
fromthesequenceidentity,theextantandancestralproteins be
.o
substantially differ in the residues present in the molecular x
fo
neighborhoodsofthepositionstargetedformutation(nearly rd
half the residues, on average, within a sphere of radius 6A˚ jou
rn
aroundeachposition;seefig.2C). als
.o
The21variantsofLBCAthioredoxin(supplementarytable rg
S1,SupplementaryMaterialonline)requiredtocalculatethe a/
t G
stabilityimpactofthetargetedmutationswerepreparedand e
o
theirthermaldenaturationwasexhaustivelycharacterizedby rg
ia
differentialscanningcalorimetry(DSC).Forallvariants,exper- In
s
iments at different protein concentrations (supplementary titu
table S2 and fig. S1, Supplementary Material online) were te o
performedtoruleoutthepossibilityofassociationequilibria. FIG. 3. Comparison of mutation effects in E. coli thioredoxin versus f T
e
LBCA thioredoxin. (A) Stability effects of mutations on LBCA thiore- c
Additionalexperimentswereperformedtoassesstherevers- h
n
doxinversusthecorrespondingeffectsonE.colithioredoxin.Mutational o
ibilityofthedenaturationprocessandscan-rateeffectonthe lo
effectsonunfoldingfreeenergies((cid:3)(cid:3)Gvalues)anddenaturationtem- g
denaturation process (supplementary table S3 and fig. S1, peratures((cid:3)T values)arecalculatedintheE.colidirection.Thecolor y o
Supplementary Material online). These studies and the code (the samme as that used in fig. 2B and supplementary table S1, n M
subsequent data analyses (see supplementary data, Supplementary Material online) refers to the relation between the arc
h
Supplementary Material online) support that the thermal mutationsactuallyperformedinthetwobackgrounds.Typicalexperi- 1
1
denaturation of the LBCA thioredoxin variants conforms to mental scatter is smaller than the size of the points. (B) Mutational , 2
0
a two-state equilibrium unfolding with some kinetic distor- effectsonE.colithioredoxinstabilityminusthecorrespondingeffectson 16
tions at temperatures higher than the measured T . Such LBCAthioredoxinstability((cid:3)(cid:3)(cid:3)Gand(cid:3)(cid:3)T values).
m m
distortion precludes the determination of reliable values for
theunfoldingheatcapacitychange((cid:3)C )and,consequently,
P
preventsusfromcalculatingmutationaleffectsonunfolding is likely linked to kinetic stability and both (cid:3)(cid:3)G and (cid:3)Tm
free energy ((cid:3)(cid:3)G values) on the basis of the integrated canbeviewedasmetricsofthemutationaleffectsonkinetic
Gibbs–Helmholtz equation. Nevertheless, since mutational stability(seeGodoy-Ruizetal.2006fordetails).
effects on denaturation temperature ((cid:3)T values) are Aplotofmutationeffectsonthestabilityoftheancestral
m
small for the I/V and E/D exchanges, we could calculate LBCA thioredoxin versus the corresponding effects on the
(cid:3)(cid:3)G’s from (cid:3)T ’s using the approximate equation pro- stability of the extant E. coli thioredoxin shows a strong
m
posed by Schellman (1987) that does not require a (cid:3)C correlation(fig.3A)withaPearsoncorrelationcoefficientof
P
value.Note,however,thatthesameconclusionsarereached 0.89, a slope close tounity (1.03(cid:2)0.21) and a value for the
usingT asanempiricalmetricofstabilityanddescribingthe probabilitythatthecorrelationoccursbyrandomchanceof
m
mutational effects on stability by the (cid:3)T values; for refer- P=9(cid:3)(cid:4)10(cid:5)8. Furthermore, the correlation holds for the
m
ence, both (cid:3)(cid:3)G and (cid:3)T values are shown in figure 3. In positions in which the extant and ancestral amino acids
m
anycase,theevolutionarystabilitythresholdforthioredoxins differ(reddatapointsinfig.3A).
444
MBE
MutationalStudiesonResurrectedAncestralProteins . doi:10.1093/molbev/msu312
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
://m
b
e
.o
x
fo
rd
jo
u
rn
a
ls
.o
rg
a/
t G
e
o
rg
ia
In
s
titu
te
o
f T
e
c
h
n
FIG.4. Aminoacidenergeticpreferencesinmodernandlaboratory-resurrectedPrecambrianthioredoxins.Foreachposition,twoaminoacidsare olo
considered.Themorepreferredaminoacidisassignedapreferencevalueofzero.Thelesspreferredaminoacidisassigneda(negative)preferencevalue g
y
weqituhalthtoemmionruesptrheefecrhreadngaemininounafcoidld.inIngaflrleceaesense,rtghyeomrdoerenaptruerfaetriroendtaemminpoeraatcuidreis((cid:3)sh(cid:3)owGnainndla(cid:3)rgTemblvuaeluleest,tererss.pReecdtivceolylo)raisssoucsieadtefdortothietsleressplparceefmererendt on M
a
aminoacidwheneveritistheaminoacidpresentinthenon-mutated(wt)protein.(A)AminoacidpreferencesforseveralpositionsinE.coliandLBCA rc
h
thioredoxins.PositionsatwhichmorepreferredaminoacidsdifferbetweenE.coliandLBCAthioredoxins(ChouandFasman1974;Mitrakietal.1991; 11
Baronietal.2004)arelabeledwithwhitenumbersinsideblackcircles.Position10isnotlabeledbecausethereisnosignificantdifferenceinpreference. , 2
0
1
(B)Aminoacidpreferenceatposition90ofthioredoxinsasafunctionofgeologictime.Totalnumbersofaminoaciddifferencesbetweenconsecutive 6
nodesareshownwithincircles.
Theexcellentancestral/extantcorrelationfound,however, fortheE.coliandLBCAthioredoxins(fig.4A).Theenergetic
shouldnotbetakentoimplythatthemutationalenergetics preference scale is constructed in the following ways: (1) A
havenotchangedatalloverthecourseofbillionsofyears.In value of zero isassigned tothe energetically more preferred
fact, when calculated in the E. coli direction, most of the aminoacid(i.e.,iftheX!Ymutationisdestabilizing,Xisthe
mutations studied are more destabilizing on the modern E. energetically more preferred amino acid; if the X!Y muta-
colithioredoxinascomparedwiththeancestralbackground tionisstabilizing,Yistheenergeticallymorepreferredamino
(fig.3B).Thisresultappearsconsistentwithsomedegreeof acid) and (2) the less preferred amino acid is assigned a
evolutionaryadjustmenttotheaminoacidresiduespresent preferencevalueequaltothemutationalchangeinunfolding
in the extant protein, that is, with the evolutionary Stokes freeenergy(orthemutationalchangeindenaturationtem-
shift(Pollocketal.2012).However,theextentofevolutionary perature) associated to the replacement of the more pre-
adjustment is insufficient to erase the ancestral pattern of ferred amino acid with the less preferred one (i.e., a
energetic preferences. To make this point visually clear, we negative value in all cases). There is a good agreement be-
have prepared plots of energetic preference versus position tweenthesetsofmorepreferredaminoacidsforE.coliand
445
MBE
Rissoetal. . doi:10.1093/molbev/msu312
LBCAthioredoxins:Only3discrepanciesoutof21instances
wereobserved(positions4,60,and61;seefig.4A)andthese
corresponded to cases in which preference differences are
quite minor. In contrast, there are 7 sequence differences
betweentheextantandancestralproteinsatthe21position
studied. As such, the energetic amino acid preferences are
moreconservedthantheresiduesthemselvesoverevolution-
arytime.
Leucine Versus Lysine Preferences at Position 90 in
Thioredoxins
Theresultssummarizedintheprecedingsectionsupportthe
notion that preferences among biochemically similar amino
acids may be conserved even over long evolutionary time-
scales.Oneobviousimplicationisthatconservationofamino D
o
acid preference is likely widespread and that it can be wn
lo
expectedtoholdovershortertimescalesandalsofordissim- a
d
e
ilar amino acids. A particularly illustrative instance of the d
lattercaseisdescribedbelow. fro
m
A lysine residue is present at position 90 in the modern h
ttp
E. coli thioredoxin, while leucine is the ancestral residue at ://m
this position along the line of descent from the ancestor to b
e
the extant E. coli protein (see phylogenetic tree annotated .ox
fo
with amino acids at position 90 in fig. 5A). Specifically, the rd
leucine residue present at position 90 is inferred for thior- jou
rn
edoxinsoftheLBCA(lastbacterialcommonancestor,about a
ls
4 billion years before present) and the last common ances- .o
rg
tor of the cyanobacterial, deinococcus, and thermus groups a/
(LPBCA, about 2.5 billion years before present). Thus, we t G
e
o
preparedvariantsoftheseproteinswiththeL90Kmutation. rg
ia
Ontheotherhand,alysineresidueispresentatposition90 In
s
in the extant thioredoxin from E. coli and inferred for the titu
laboratory resurrection corresponding to the thioredoxin of te
o
the last common ancestor of (cid:2)-proteobacteria (LGPCA, f T
FIG.5. Evolutionaryhistoriesoftheaminoacidpresentatposition90in e
about 1.5 billion years before present); therefore, we pre- thioredoxins (A) and position 182 in b-lactamases (B). Phylogenetic chn
pared variants of these proteins with the K90L mutation. trees used for ancestral sequence reconstruction of thioredoxins olo
g
Wedeterminedthestabilityofthe“wild-type”(wt)proteins (Perez-Jimenez et al. 2011) and b-lactamases (Risso et al. 2013 ) are y o
andthecorrespondingmutantvariantsusingDSC.Someof color coded according to the residue present: Red (extant), blue n M
thedeterminedmutationaleffectsondenaturationtemper- (ancestral), and grey (other). Small circles are used to highlight the arc
ature were very large (up to about 15(cid:6)) and, therefore, the evolutionary trajectory from the oldest ancestor to the extant E. coli h 1
use of Schellman equation (Schellman 1987 ) to calculate protein. 1, 2
0
mutational effects of unfolding free energy ((cid:3)(cid:3)G values) 16
was not advisable in this case. As an alternative, we elected
to use denaturation temperature values as a metric for sta- 5A). There is certainly evidence of adjustment to the “new”
bility and to construct the amino acid preference scale on lysine residue, as the effect of mutation in the K!L direc-
the basis of the mutation (cid:3)T ’s. The results are summa- tion is more stabilizing for the oldest thioredoxins in which
m
rized by plotting amino acid preference against a geologic leucineistheresidue presentatposition 90(fig.4B).Thisis
timescale (fig. 4B). In all cases, mutations were found to be consistent with either preadjusting or permissive changes
stabilizing in the K!L direction (i.e., K90L was found to be (previousmutationsinthespatialneighborhoodofposition
stabilizing in the E. coli and LGPCA background and L90K 90 “permitted” the introduction of a lysine residue at posi-
wasfoundtobedestabilizingintheLPBCAandLBCAback- tion90)ortheevolutionaryStokesshift(Pollocketal.2012)
grounds; see supplementary fig.S3, Supplementary Material (energetic adaptation to the new residue after it has been
online). Therefore, the energetic preference of L over K is introduced).Theadjustment,however,doesnotchangethe
conserved over ~4 billions of years, despite the fact that, ranking of amino acid preferences at this site and replace-
according to the ancestral reconstruction, the lysine at po- mentwiththeancestralaminoacid(i.e.,themutationK90L)
sition 90 appeared about 2 billion years ago in the line of does stabilize E. coli thioredoxin (see fig. 4 and supplemen-
descent leading to the extant E. coli protein (figs. 4B and tary fig. S3, Supplementary Material online).
446
MBE
MutationalStudiesonResurrectedAncestralProteins . doi:10.1093/molbev/msu312
antibioticresistance.Themutationisknowntobestabilizing
intheextantTEM-1backgroundandthisstabilizingeffecthas
been proposed to permit the acquisition of destabilizing
mutations that enhance catalytic efficiency toward a new
antibiotic (Huang and Palzkill 1997; Wang et al. 2002;
Bloom et al. 2005; Salverda et al. 2010). Reconstruction of
ancestral lactamase sequences (Risso, Gavira, Gaucher, et al.
2014)supportsthatthemethionineresidueatposition182in
TEM-1 b-lactamase appeared comparatively recently in the
lineofdescentleadingtotheextantTEM-1protein(figs.5B
and 6 and supplementary fig. S4, Supplementary Material
online), while a threonine is present in sequences of many
modern b-lactamases and also in the reconstructed
sequences corresponding to the last common ancestors of
(cid:2)-proteobacteria (GPBCA, about 1.5 billion years before
D
present), various Gram-negative bacteria (GNCA, about 2 o
w
billion years before present), andvarious Gram-positive and nlo
a
Gram-negative bacteria (PNCA, about 3 billion years before d
e
d
present)(fig.6A).Wethuspreparedtheproteinsencodedby fro
thesereconstructedsequenceswithandwithouttheT182M m
h
mutation,whiletheextantTEM-1b-lactamasewasprepared ttp
with and without the original global suppressor mutation ://m
b
M182T (see supplementary data, Supplementary Material e
.o
online,fordetails).DSCstudiesonthethermaldenaturation xfo
ofallthesewtandvariantformsshowedthattheenergetic/ rdjo
structuralpreferenceofToverMisconservedoverbillionsof urn
a
years(fig.6B).Thatis,themutationT182Mintheancestral ls
.o
backgroundsisdestabilizingandthemutationM182Tinthe rg
extant TEM-1 background is stabilizing (supplementary fig. at G/
S4,SupplementaryMaterialonline).Noclearevidenceofad- eo
justmenttothenewresidueisapparentinthiscase.Thisis rgia
perhaps due to the inference that a methionine at position In
s
182 appeared only recently in the evolutionary trajectory titu
te
leadingtotheTEM-1b-lactamase(figs.5Band6).Wehave o
alsostudiedtheeffectoftheM/Texchangeonthestabilityof f Te
c
theextantb-lactamasefromB.licheniformis(figs.5Band6A). hn
FIG. 6. Amino acid energetic preferences at position 182 for modern Inthiscase,athreonineresidueispresentinthewtprotein olo
and laboratory-resurrected Precambrian b-lactamases (see main text gy
and,therefore,wepreparedtheb-lactamasefromB.licheni- o
and legend to fig. 4 for a description of the preference scale). (A) n
formis with and without the T182M mutation. The corre- M
Schematic phylogenetic tree showing b-lactamase nodes relevant to a
this work (see fig. 1 for definitions). Numbers alongside the nodes sponding scanning calorimetry profiles are compared with rch
standforthevaluesofthesequenceidentitywiththeextantprotein. thosefortheTEM-1lactamasein figure6C.M182Tisstabi- 11
(B)Aminoacidpreferencesatposition182inb-lactamasesasafunction lizingintheTEM-1background,whileT182Misdestabilizing , 20
1
ofgeologictime.Thedatapointat“today” correspondstotheE.coli intheB.licheniformisbackground.Thatis,inbothinstances 6
TEM-1 lactamase. Total numbers of amino acid differences between the mutation is stabilizing in the M!T direction, further
consecutivenodesareshownwithincircles.(C)EffectoftheM/Tex- supporting the conservation of energetic preference. It is to
changeatposition182onthestabilityoftwoextantb-lactamasesas
benotedthatTEM-1b-lactamaseandtheb-lactamasefrom
followedbyDSC.Amethionineispresentatposition182inE.coliTEM-
B. licheniformisshare a common ancestoron the orderof 3
1 lactamase, while threonine is the residue at that position in the
billion years ago (figs. 5B and 6A). These extant proteins,
lactamasefromBacilluslicheniformis.M182TisstabilizingintheTEM-
therefore, may be viewed as being separated by ~6 billion
1backgroundandT182MisdestabilizingintheB.licheniformisback-
yearsofevolutionand,infact,theyshowonly37%sequence
ground.Inbothcases,themutationisstabilizingintheM!Tdirection,
indicatingpreferenceconservation. identity.
A Plausible Evolutionary Explanation for the
Methionine Versus Threonine Preferences at Position Occurrence and Persistence of Less Preferred Amino
182 in b-Lactamases Acids
The global suppressor M182T mutation appears often in A rather noteworthy result reported in this work is the
TEM-1b-lactamaseslinkedtoclinicalcasesofemergenceof observation that billion-year-old conservation of energetic
447
MBE
Rissoetal. . doi:10.1093/molbev/msu312
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
://m
b
e
.o
x
FIG.7. Evolutionaryhistoryoftheaminoacidoccupancyatseveralpositionsinthioredoxinsandb-lactamasesasinferredfromancestralsequence fo
reconstruction.Inallthepositionsshown,theaminoacidpresentintheextantprotein(the“extantaminoacid”)isnottheenergeticallymorepreferred. rdjo
u
Extantaminoacidsareshowninredcolor,insuchawaythatthetimeinwhichtheyappearedisvisuallyapparent.Twoconsecutiveone-letteramino rn
a
acidcodesareusedwhendifferentaminoacidsarepresentattwonodesofverysimilarage(ifthesameaminoacidispresentattwonodesofsimilar ls
.o
age,asingleletterisshown).Valuesofsequenceidentitywiththeextantproteinareshownforthesequencesinvolvedintheancestraltoextantamino rg
acidtransition.(seesupplementaryfig.S2,SupplementaryMaterialonlineforfurtherdetails). a/
t G
e
o
rg
preferenceexistsatpositionsinwhichtheaminoacidpresent present (on the order of billions of years in most cases). As ia In
has changed in the line of descent leading from the oldest previouslypointedout,however,thestabilityofnaturalpro- stitu
ancestortotheextantprotein.Wefoundsixexamplesofthis teinsismarginal(TavernaandGoldstein2002;Bershteinetal. te
o
scenario.Theyaresummarizedinfigure7wherethecommon 2006;Godoy-Ruizetal.2006;Bloometal.2007;Tokurikietal. f T
patternisapparent.Theresiduepresentintheextantprotein 2007;Sikosek and Chan 2014), that is, just slightly above an ec
h
n
(the “extant” amino acid) differs from the residue in the evolutionarystabilitythreshold,and,thus,evenamoderately o
lo
oldest ancestral protein (the “ancestral” amino acid) but destabilizingmutationcouldpotentiallyimpairproperfolding gy
o
the energetic preference for the ancestral amino acid over or facilitate degradation with a subsequent deleterious n
M
theextantoneisconserved.Ancestralsequencereconstruc- impactonorganismalfitness.Accordingtoastabilitythresh- a
rc
tion provides estimates of the geologic time at which the old selection scenario, destabilizing mutations are still often h 1
1
extantaminoacidfirstappearedinthelineofdescentleading accepted(otherwise,proteinstabilitywouldnotbemarginal). , 2
0
totheextantprotein(seefig.7andannotatedphylogenetic Acceptance of a mutation with a destabilizing effect may 1
6
trees in fig. 5 and supplementary figs. S2 and S4, require that a previous stabilizing mutation (i.e., a compen-
Supplementary Material online). Such times range from sating or permissive mutation) pave the way for the accep-
severalhundredmillionyears(forthemethionineresidueat tanceofthedestabilizingone(Weinreichetal.2006;Bloom
position182inb-lactamase)toabout2billionyears(forthe etal.2007;Ortlundetal.2007;Wyganowskietal.2013).Itis
aspartateresidueatposition43andthelysineresidueatpo- importanttonoteinthiscontextthatproteinstabilitythresh-
sition 90 in thioredoxins) and correspond to differences in olds are unlikely to remain constant during evolution.
sequenceidentitythatrangebetween0.59and0.69. Actually,severalthreshold-relaxingeventsmayhaveplausibly
Overall, the six cases collected in figure 7 provide clear occurred and facilitated the acceptance of destabilizing
evidence that energetic preferences may be conserved over mutations: (1) The development of efficient chaperone
planetary timescales even when the amino acid residues systems may lower stability thresholds, as suggested by the
themselves change during evolution. However, they also fact that chaperonin overexpression can promote enzyme
pose some obvious evolutionary questions that need to be evolutionbyallowingthefoldingofvariantswithfunctionally
addressed. It follows from the preference conservation that usefulbutdestabilizingmutations(TokurikiandTawfik2009;
the mutation to the extant (less preferred) amino acid was Wyganowskietal.2013);(2)accordingtooneproposal,the
destabilizing when it occurred at a particular time before temperatureoftheoceanshasdecreasedoverbillionsofyears
448
MBE
MutationalStudiesonResurrectedAncestralProteins . doi:10.1093/molbev/msu312
FIG.8. EffectoftheK90LmutationinthioredoxinonE.colifitness.Theresultsofwt/K90Lcompetitionexperimentsinbatchculturesforlongperiodsof
timeareshown.Twenty-threeindependentexperimentsweresetupandthefractionsofcellscontainingwt(red)andK90L(blue)thioredoxinswere
determinedafter5and15days,respectively.Notethatin22(outof23)experiments,cellscontainingwtthioredoxin“win”thecompetitionafter15days.
D
o
w
n
(KnauthandLowe2003;Gaucheretal.2008),thusproviding competition experiments in batch culture for long periods loa
d
ample opportunities to relax the stability threshold for of time (about 2 weeks) without addition of nutrients. The ed
ancientlifelivinginancientoceans;(3)organismalmigration rationalebehindthisapproachisthatconditionsinlong-term fro
m
from a high-temperature local environment (hydrothermal stationary-phasecultureshavebeenproposedtomimiccon- h
systems for instance; Lane and Martin 2013) to more ditions found in natural environments (Finkel 2006). Briefly, ttp://m
temperateenvironmentcouldalsolowerthestabilitythresh- wesetuppaircompetitionexperimentsofasinglecloneof b
e
old for the proteins of the migrating organisms; and (4) thestraincomplementedwithwtthioredoxinversusasingle .o
x
comparatively short periods of sharp decreases in planetary cloneofthestraincomplementedwiththeK90Lvariant.The ford
temperatureareknowntohaveoccurred(globalglaciations, proportionsofthetwovariantsineachpopulationwerede- jou
usually referred to as Snowball Earths; Hoffman et al. 1998; terminedat5and15daysafterthestartofthecompetition rna
ls
Kirschvinketal.2005)andmayhavepossiblyfacilitatedthe usingSangersequencingandtheQSVanalyzerprogram(Carr .o
rg
occurrenceofsomehighlydestabilizingmutations. etal.2009),amethodologythatdoesnotrequiretheuseof a/
Overall,itisclearthatseveralplausiblescenariosmayex- markers(whichcouldpotentiallyhaveaneffectonfitness).In t G
e
plain why proteins accumulate less energetically preferred order to rule out the possibility that the “winner” of the org
aminoacids.However,somespecificexplanationisrequired competition is determined by fitness differences between ia In
forthefactthatthese“energeticallysub-optimal”aminoacids clones that are not related to the K90L mutation in thiore- stitu
persist over significant evolutionary periods, since estimates doxin, we performed 23 independent competition experi- te
o
of neutral mutation rates (Ochman et al. 1999) obviously ments (i.e., with 23 independent pairs of clones). After 5 f T
predict that mutational changes will occur over the billion days of competition, wt/K90L population ratios for the 23 ec
h
n
yearstimescale of figure 7.The simplestexplanation forthe experiments showed some dispersion although the average o
lo
evolutionarypersistenceoflesspreferredaminoacidsisthat valuewasclosetounity,indicatingnosystematicbias(fig.8). gy
they allow protein functional properties that lead to Ontheotherhand,after15days22(outof23)competition on
M
enhanced organismal fitness. In order to obtain some experiments displayed a wt/K90L population ratio higher a
rc
experimental insights into this scenario, we have consid- than unity, indicating a clear preference of the “wt strain” h 1
ered the functional impact of the K/L exchange in over the “K90L” strain (fig. 8). The interpretation of these 1, 2
thioredoxins—proteinsthatregulatemanycellularprocesses organismalfitnessexperimentsiscomplex.Onepossibilityis 01
6
(Holmgren1985)andthatproteomicanalyses(Kumaretal. thatthegeneticalterationsleadingtothegrowthadvantage
2004) have identified as having a large number of protein in the stationary phase phenotype (Finkel 2006) are more
binding partners in vivo. It is clear, therefore, that activity probabletooccurinthewtstrain,thusamplifyinganorigi-
assays in vitro are of limited usefulness in this context, as nally small difference in fitness. This notwithstanding, the
fitness experiments summarized in figure 8 are consistent
they cannot provide information about the effect of the
withanevolutionarynarrativethatinvolvesacceptanceofa
K/L exchange on the multitude of biomolecular processes/
destabilizing mutation linked to relaxation of the stability
interactions in which thioredoxin participates in vivo.
threshold and persistence of the less preferred amino acid
Consequently, we elected to directly measure the effect of
because of functional advantages that translate into en-
theK/Lexchangeatposition90ofthioredoxinsonorganismal
hancedorganismfitness.
fitness. It is obviously difficult to perform fitness studies on
Precambrian micro-organisms, but the effects of these
On the Molecular Basis of Amino Acid Preference
proteins on modern organisms can be determined. We
thus complemented a thioredoxin-deficient E. coli strain Conservation
with plasmids containing either the wt thioredoxin gene or The experimental results reported in this work show that
the gene carrying the K90L mutation. We performed amino acid preferences in proteins can be conserved over
449