Diversification of a single ancestral gene into a successful toxin superfamily in highly venomous

16 Pages·2014·1.45 MB·English

by  Sandy S Pineda

Pinedaetal.BMCGenomics2014,15:177
http://www.biomedcentral.com/1471-2164/15/177
RESEARCH ARTICLE Open Access
Diversification of a single ancestral gene into a
successful toxin superfamily in highly venomous
Australian funnel-web spiders
Sandy S Pineda1†, Brianna L Sollod2,7†, David Wilson1,3,8†, Aaron Darling1,9, Kartik Sunagar4,5, Eivind A B Undheim1,6,
Laurence Kely6, Agostinho Antunes4,5, Bryan G Fry1,6* and Glenn F King1*
Abstract
Background: Spiders have evolved pharmacologically complexvenomsthatserve to rapidly subdue preyand
deter predators.The major toxic factors in most spider venomsare small, disulfide-rich peptides. While thereis
abundant evidence that snake venoms evolved by recruitmentof genes encoding normal body proteins followed
byextensive gene duplication accompaniedby explosivestructural and functional diversification, the evolutionary
trajectory of spider-venom peptides is less clear.
Results: Here wepresent evidence of a spider-toxin superfamily encoding a high degree ofsequenceand
functional diversity thathas evolved via accelerated duplication and diversification ofa single ancestral gene. The
peptideswithinthistoxinsuperfamilyaretranslated asprepropeptides thatareposttranslationallyprocessedtoyield
thematuretoxin. The N-terminal signal sequence, as well as the protease recognition site at thejunction ofthe
propeptide and mature toxin are conserved,whereastheremainder of the propeptide and mature toxin sequences
are variable. Alltoxin transcriptswithin this superfamily exhibit a striking cysteine codon bias. We show that
different pharmacologicalclasses of toxins within this peptide superfamily evolved under different evolutionary
selection pressures.
Conclusions: Overall, this study reinforces thehypothesis that spiders use a combinatorial peptide library strategy
to evolve a complex cocktailof peptide toxins thattarget neuronalreceptorsand ion channels inpreyand
predators. We show that theω-hexatoxins that target insect voltage-gated calcium channels evolved under the
influence of positive Darwinian selection inan episodic fashion, whereas theκ-hexatoxinsthat targetinsect
calcium-activated potassium channels appear to be under negative selection. A majorityof thediversifying sites in
theω-hexatoxins are concentrated onthe molecular surface ofthe toxins, thereby facilitating neofunctionalisation
leading to new toxin pharmacology.
Keywords: Spider toxin, Spider venom, Hexatoxin, ω-hexatoxin,κ-hexatoxin,Australian funnel-web spider, Molecular
evolution, Gene duplication, Positiveselection,Negative selection
*Correspondence:[email protected];[email protected]
†Equalcontributors
1InstituteforMolecularBioscience,TheUniversityofQueensland,306
CarmodyRoad,StLucia,QLD4072,Australia
6VenomEvolutionLab,SchoolofBiologicalSciences,TheUniversityof
Queensland,StLucia,QLD4072,Australia
Fulllistofauthorinformationisavailableattheendofthearticle
©2014Pinedaetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
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reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain
Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,
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Background producelargemultigenefamilies.Thisprocessisanalogous
Venomshaveproventobekeyevolutionaryinnovations to the birth-and-death model of evolution proposed for
for many divergent animal lineages [1,2]. Although the multigene families involved in adaptive immunity, such as
mostextensivelystudied venoms arefromthemedically themajorhistocompatibilitycomplexandimmunoglobulin
important scorpions, snakes, and spiders, venom sys- V genes [6]. However, the evolutionary trajectory is less
H
tems are present in many other lineages including clear for the venoms of spiders, scorpions, and molluscs,
cnidarians, echinoderms, molluscs, fish, lizards, and which are dominated by disulfide-rich peptides of mass
mammals [1,2]. These venoms have evolved to serve a 2–9kDa[7-12].Thesepeptidestypicallypossesshighaffin-
variety of purposes, including prey capture, competitor ity and often-exquisite specificity for particular classes of
deterrence, and defense against predators. There has ion channels and other nervous system targets [13-15].
been considerable innovation both in the chemical These neurotoxic functions are perhaps not surprising
composition of these venoms as well as the method of given that the primary role of these venoms is to paralyse
venom delivery, which includes barbs, beaks, fangs, orkillenvenomatedprey[11,16,17].
harpoons, nematocysts, pinchers, proboscises, spines, In this study, we analysed toxin-encoding transcripts
spurs,andstingers[1,2]. from five species of Australian funnel-web spider (Aranae:
From a molecular evolutionary perspective, the venoms Mygalomorphae:Hexathelidae:Atracinae)fromthegenera
of snakes are the best understood. There is now abundant Atrax and Hadronyche, representing a geographic spread
evidence that snake venoms evolved by recruitment of of more than 2000 km (Figure 1), in order to provide
genes encoding normal body proteins followed by exten- insightintotheevolutionarytrajectoryoftheω-hexatoxin-
sive duplication, neofunctionalization, and in some in- 1 (ω-HXTX-1) family. ω-Hexatoxins (formerly known as
stances relegation to the status of pseudogene [1,3-5]. In ω-atracotoxins) are peptides comprising ~37 residues that
manycases,thesegeneshavebeenexplosivelyreplicatedto were first isolated from the venom of the lethal Blue
Figure1Distribution,venomcollectionandvenom-glanddissectionofAustralianfunnel-webspiderspeciesusedinthisstudy.(A)
MapoftheeasternhalfofAustraliashowingthedistributionofthefivespeciesofAustralianfunnel-webspiderusedinthisstudy.(B)Female
funnel-webspider(Hadronycheinfensa)fromFraserIsland,QLD.Inresponsetoprovocation,thespiderhasadoptedatypicalaggressive/defensive
posture,withfrontlegsandpedipalpsraisedandthefangsinanelevatedpositionreadytostrike.Notethedropofvenomoneachofthefang
tips.(C)AsingleH.versutavenomglandthathasbeendissectedfromthesurroundingmuscletissue.Thevenomglandintheseandother
mygalomorphspidersislocateddirectlybelowthedorsalsurfaceofthechelicerae.
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Mountains funnel-web spider Hadronyche versuta [18]. Results and discussion
The ω-hexatoxins are major components in the venom of Theω-hexatoxinsareexpressedasprepropeptide
Australian funnel-web spiders [18-20] and they contribute precursors
significantlytopreyimmobilizationbyvirtueoftheirability RACE analysis was used to amplify transcripts encoding
to specifically block insect, but not vertebrate, voltage- orthologsofω-HXTX-Hv1afromfourspeciesofAustralian
gated calcium (Ca ) channels [17,18,20-22]. Their po- funnel-webspider:Atraxrobustus,H.infensa,H.venenata,
V
tentinsecticidalactivityhasengenderedinterestinthese and H. versuta (Figure 2). Multiple ω-HXTX-Hv1a ortho-
peptides as bioinsecticides [11,17,23]. Proteomic analysis logswereidentifiedineachspecies(i.e.,24paralogsencod-
ofH.versutavenomrevealedanumberofω-HXTX-Hv1a ing seven distinct mature toxins were identified in H.
paralogs [19],suggestingthatthispeptide toxin mightbe- infensa,18paralogsencodingsixmaturetoxinswereidenti-
long to a multigene family. However, because the venom fiedintheSydneyfunnel-webspiderA.robustus,andeight
used in this previousstudywaspooled from several spi- paralogs encoding two mature toxins identified in the
ders,it wasunclearwhethertheseapparentparalogsare Tasmanian funnel-web spider H. venenata) (Figure 3A).
simply polymorphisms resulting from allelic variation. A further eight paralogs encoding four distinct mature
By using cDNA libraries obtained from a single spider, toxins were identified in the venom-gland transcriptome
we demonstrate here that ω-HXTX-Hv1a is indeed part ofH.modesta(Figure3A).Thus,theaminoacidsequence
of a large multigene family that appears to have arisen diversitypreviouslyreportedforω-HXTX-1basedonana-
from explosive gene duplication followed by extensive lysisofpooledvenomsamples[19]isduetoexpressionof
sequence divergence and neofunctionalization. Within multiple related transcripts in a single spider rather than
thissuperfamily of toxins, weshowthat pharmacologic- allelic variation. The almost complete conservation of the
ally distinct toxin classes are evolving under starkly signalsequence,aswellasthepatternofconservedcyste-
different selection pressures, with some toxin classes ines in the mature toxin (Figure 3A), indicates that these
accumulating variation under episodic bursts of adapta- ω-HXTX-Hv1a homologs arose by duplication and se-
tion, while others remain constrained by negative selec- quencedivergenceoftheoriginaltoxin-encodinggene.
tion. This work reinforces the idea that the chemical All of the ω-HXTXs are expressed as prepropeptide
and pharmacological diversity present in spider venoms precursors that are posttranslationally processed to yield
may have evolved from a relatively small number of an- thematuretoxinsequence(Figure2A).Thehighlyhydro-
cestralgenes. phobic 22-residue signal sequence is of similar length to
Figure2Schematicrepresentationoftoxinprecursors,overallRACEamplificationstrategyandidentificationoftoxinsuperfamilies.
(A)Schematicrepresentationofatypicalspiderpeptideprecursorshowingthesignalpeptideinorange,thepropeptideinpurple,andthe
maturetoxininblack.Aftertranslation,thesignalandpropeptideregionsareproteolyticallyremovedtoyieldafunctionalmaturetoxin.(B)
GeneraloverviewoftheRACEprotocolforsequencinghexatoxintranscripts.Adaptorsareaddedtothe5’and3’endoftranscriptsduringcDNA
librarypreparation.Inboth3’and5’RACE,gene-specificprimersareusedintheforward(3’RACE)orreverse(5’RACE)orientationtoamplify
full-lengthsequences.TheresultingPCRproductsarethenclonedandsequenced.(C)SchematicrepresentationoftheShivasuperfamily
highlightingthecombinatorialnatureofspider-venompeptides.
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Figure3Sequencealignmentsofωandκ-hexatoxins.(A)Sequencealignmentofω-HXTX-Hv1aparalogsfromeachspecies:Hadronyche
modesta(Hmo1a–Hmo1d),Atraxrobustus(Ar1a–Ar1g),Hadronycheinfensa(Hi1a–Hi1g),andHadronychevenenata(Hvn1aandHvn1b).Thelevelof
residueconservationisgradedfromblack(fullyconservedacrossallparalogs)todarkgrey(conservedinmosttoxins)tolightgrey(conservedin
amajorityoforthologs).Thelinesbelowthesequencealignmentindicatethedisulfide-bondconnectivityofω-HXTX-Hv1a.(B)Alignmentof
κ-HXTX-Hv1aparalogsfromHadronycheversutaandoneorthologfromHadronychemodesta.Thelevelofresidueconservationisgradedasin
panel(A)andthesignalpeptide,propeptide,andmaturetoxinregionsarehighlighted.Thelinesbelowthesequencealignmentindicate
thedisulfide-bondconnectivityofκ-HXTX-Hv1c,withthevicinaldisulfidebondhighlightedinred.Notethatω-HXTX-Ar1a(UniProtPF06357),
ω-HXTX-Hi1a(UniProtP0C2L5),ω-HXTX-Hi1b(UniProtP0C2L6),ω-HXTX-Hi1c(UniProtP0C2L7),andκ-HXTX-Hv1c(UniProtP82228)havebeen
previouslyisolateddirectlyfromvenom.
that reported for peptide-toxin precursors from spiders, study. These species are distributed along the eastern
scorpions, and cone snails [8]. The 20-residue propeptide seaboard of Australia with a geographic spread of more
sequenceishighlyacidic,withanetchargeof−4,afeature than 2000 km (Figure 1). The hexathelids are a group of
thathasbeennotedfornumerousspider-toxinpropeptide approximately40speciesdividedintothreegenera:Atrax,
sequences [24-28] but which is not characteristic of toxin Hadronyche and Illawarra [34-36]. They are adapted to
precursors from other venomous animals. Moreover, forestenvironmentsbutcanalsobefoundinhabitatsthat
the presence of a propeptide regioncontrasts with most range from montane herblands and open woodland to
scorpion-toxin precursors in which the signal sequence closedforest[36].Conservationoftheω-hexatoxinfamily
is fused directly to the mature toxin without an inter- oftoxinsoverthiswiderangeofenvironmentsanddiffer-
vening propeptide [8]. The reason for the highly acidic ing prey distributions implies that there has been strong
propeptide region in spider toxin precursors remains to evolutionarypressuretomaintainthesepeptidesaspartof
be determined, but it may be related to specific interac- the venom arsenal, which is perhaps not surprising given
tions between the toxin precursor and components of thattheyarebroadlyactiveagainstmanydifferentarthro-
the secretory and/or protein folding pathway in spider pods[11,17,37].
venomglands. In addition to obvious homologs of ω-HXTX-Hv1a,
ThepropeptidesequenceterminateswithadibasicArg- the RACE and transcriptomic analyses revealed add-
Arg signature;dibasicsequencesare commonrecognition itional families of toxins that had almost identical signal
sites for proteolytic removal of propeptide segments in sequences to the ω-HXTX-1 transcripts, but divergent
neuropeptide precursors from both vertebrates [29] and propeptide and mature toxin sequences. We named one
invertebrates [30]. While Arg is the terminal residue in of these families the ω/κ-HXTX family (described as U-
virtuallyall knownspider-toxinpropeptidesequences,the ACTXin[38]).Theω/κ-HXTXpeptidesappeartobedis-
penultimate residue is variable, though it is commonly tributed in two of the species examined (A. robustus and
Asp,Glu,orLys[25,26,31-33]. H.versuta);thisreinforcestheideathatthesetoxinsmost
likely arose ancestrally by duplication of a ω-HXTX-1
ω-hexatoxinsbelongtoalargetoxin-genesuperfamily gene followed by hypermutation of the propeptide and
Orthologs of ω-HXTX-Hv1a were identified in all five mature-toxin regions in order to create a new function
species of Australian funnel-web spider examined in this (neofunctionalization). The conservation and radiation of
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these toxinsacrossthis family of spiders implies that they signal peptide, propeptide, and mature toxin [8]. A re-
are not nonfunctional relics of an explosive radiation of vised logo analysis of the Shiva superfamily (Figure 4A)
this toxin-gene superfamily, and we confirmed this by that incorporated all of the new sequences and species
showing that recombinant ω/κ-HXTX-Hv1a is highly reported here reinforced the dichotomy in evolutionary
insecticidal [38]. The high insecticidal potency of this forces affecting various elements of the toxin precursor.
familyofpeptidesisbelievedtoresultfromasynergistic The signal peptide has clearly been highly conserved
effect on insect voltage-gated calcium (Ca ) channels throughouttheevolutionofthistoxinsuperfamilyanditis
V
andcalcium-activatedpotassium(K )channels[38]. presumably under negative selection in order to ensure
Ca
RACE analysis of the venom-gland cDNA library from that these toxins are directed to the appropriate secretory
H.versutaalsoledtoamplificationoftranscriptsencoding pathway.Incontrast,thereissignificantsequencevariation
the insecticidal toxin κ-HXTX-Hv1c [39], and sequencing in both the propeptide and mature toxin sequences, with
of the venom-gland transcriptome from H. modesta also two notable exceptions. First, in contrast to the highly
uncovered an ortholog of this toxin (Figure 3B). This was variable upstream region of the propeptide sequence, the
entirely unexpected since this toxin has a vastly differ- C-terminal proteolytic recognition signal (Arg-Arg) is
ent primary structure to ω-HXTX-Hv1a [39]. Moreover, completely preserved (Figure 4A). Presumably there has
in addition to the six conserved cysteine residues in ω- been strong selection pressure to ensure processing of the
HXTX-Hv1a that form an inhibitor cystine knot (ICK) propeptide by a specific protease. Second, in contrast to
motif [40,41], κ-HXTX-Hv1c contains two additional theoveralllowlevelofconservationofthematuretoxin
cysteineresiduesthatformanextremelyrarevicinaldisul- sequence, the cysteine residues, which direct the three-
fide bond [42-44]. Furthermore, in contrast to ω-HXTX- dimensional(3D)foldofthetoxins,are completelycon-
Hv1a,whichblocksinsectCa channels,κ-HXTX-Hv1cis served (Figure 4A). The marked variation in levels of
V
apotentandspecificblockerofK channels[45].Never- sequence conservation between the spider-toxin signal
Ca
theless, the near identity of the signal sequence in these sequence and the propeptide and mature toxin regions
two toxin families and the conservation of cysteine resi- isreminiscentofthatobservedforsuperfamiliesofcone
dues in the mature toxin indicate that they evolved from snailtoxins[46-51].
the same ancestral toxin gene and are members of the There are two striking differences between the Shiva
samegenesuperfamily. superfamily precursors and transcripts encoding human
We did not find orthologs of κ-HXTX-Hv1c in any of neuropeptides and other secreted proteins. First, whereas
theotherthreespeciesofAustralianfunnel-webspider(H. precursorsofhumanneuropeptidesoftenencodemultiple
infensa,A.robustus,andH.venenata).However,κ-HXTX- mature neuropeptide sequences [29,52], we and others
Hv1a,κ-HXTX-Hv1b,andκ-HXTX-Hv1careexpressedat have not found any examples of spider-toxin transcripts
very low levels in H. versuta venom [42], and conse- that encode more than a single mature toxin sequence.
quently we cannot rule out the possibility that these Secondly, in direct contrast to the toxin precursors, the
toxins are present in the venom of the other three spi- sequence of the mature human neuropeptide(s) is usually
ders but the transcript levels are too low to be detected strongly conserved whereas there is significantly more
usingthemethodsemployedhere. variability inthe signalsequence. This isperhaps not sur-
prising given that human neuropeptides usually act on a
TheShivasuperfamilyofpeptidetoxins singlewell-definedmoleculartargetwhereasspidertoxins
It has previously been suggested that superfamilies of typically targeta specificsubtype of receptor orionchan-
spider-venom peptides evolved from a single ancestral nel that nevertheless might vary significantly in primary
gene via explosive gene duplication [8]; the work de- structure between prey taxa (Note that most spiders are
scribed here further supports this idea as it is clear that generalist predators that target a phylogenetically diverse
the ω-HXTXs, ω/κ-HXTXs, and κ-HXTXs belong to a range of prey). Thus, expressing a family of related toxins
large superfamily of toxins that arose via gene duplica- in the venom (essentially a mini-combinatorial peptide li-
tion (Figure 2C). We have chosen to name spider-toxin brary) might ensurethat the desired receptor/ion channel
gene superfamilies after deities of death and destruction istargeted,regardlessofpreytaxa.
since the major biological role of these toxins is to
paralyze and/or kill envenomated prey. Accordingly, we Position-specificcysteinecodonbias
have named the ω/κ-HXTX/ω-HXTX/κ-HXTX gene Mature ω-HXTXs contain three disulfide bonds with 1–
superfamily after the Hindu deity Shiva, commonly 4, 2–5, 3–6 connectivity. These disulfides form an ICK
knownasthe“destroyer”. motif that provides these toxins with a high degree of
Sequence logos were previously used to analyse differ- chemical, thermal and biological stability [53]. Although
ences in the level of sequence conservation between the it is clear from a protein structure viewpoint why these
three parts of the ω-HXTX toxin precursor, namely the six cysteine residues need to be strictly maintained in
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Figure4SequencelogoandcodonusageanalysisfromtheShivasuperfamily.(A)Sequencelogo[54]basedonalignmentof
prepropeptidesfromtheShivasuperfamily.Thereisamuchhigherlevelofsequenceconservationwithinthesignalpeptidethanwithinthe
propeptideandmaturetoxinregions.Note,however,thatthecysteineresiduesthatformthecystine-knotmotifinthematuretoxinandthe
Arg-Argproteaserecognitionsitethatterminatesthepropeptideregionarebothcompletelyconserved(highlightedinblueandred,
respectively).(B)Codonusageforthesix-cysteineresiduesthatformthecystine-knotmotif.NotethestrongbiasforTGCatcysteinepositions1,
3,4,and6.Shownabovethehistogramisthedisulfidebridgearrangementforthesixcysteinesasinferredfromthe3Dstructuresof
ω-HXTX-Hv1aandκ-HXTX-Hv1c.
order to preserve the toxin’s 3D scaffold, one would not disulfidebond(Figure4B).Theobserved position-specific
expecttofindapreference foreitheroneofthetwopos- codon bias is not simply a manifestation of global codon
sible cysteine codons (TGT and TGC). Intriguingly, bias in thesespidersas wehaveobserved a preferencefor
however, previous analysis of ω-HXTX precursors re- TGT as opposed to TGC for cysteine residues in other
vealed a strong bias for TGC at four of the six cysteine hexatoxin superfamilies (data not shown). Moreover, we
positions in the mature toxin region [8]. An extended did not observe extreme codon bias for any other con-
logoanalysis[54]incorporatingallofthenewlydiscovered servedresidueinthematurehexatoxins.
sequences reported in this study corroborated the previ- Position-specific cysteine-codon bias has also been ob-
ously observed codon bias (Figure 4B). We found an ex- served in superfamilies of cone snail toxins and it has
tremeTGC codon bias for the four cysteine residues that been proposed that these codons might serve as attrac-
form the 1–4 and 3–6 disulfide bridges in the ω-HXTX tants for a mutator complex that includes a poorly pro-
family but not for the two cysteines that form the 2–5 cessive and highly mutagenic polymerase (e.g., DNA Pol
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V) that promotes radiation of the toxin superfamily by site-specific models (Table 1: codon numbers based on κ-
facilitating hypermutation of the mature toxin region HXTX-Hv1c_2 and ω-HXTX-Ar1a_1; Additional file 1:
[49,50]. However, there is currently no direct evidence Table S1–3). Model 8 estimated ω of 0.69, 1.06 and 0.78
that cysteine-codon bias plays a part in directing the for the ω-HXTXs, κ-HXTXs, and the combined Shiva
evolutionofspiderorconesnailtoxins. superfamily dataset, respectively (Table 2 and Additional
file 1: Table S1–3). Although the computed ω for the
Molecularevolutionanalyses κ-HXTXs was >1, the assessment was not statistically
We utilized various state-of-art molecular evolutionary significant (p>0.05) in comparison with the null model
assessment methods to determine the influence of nat- (M7β).TheBayesEmpiricalBayes(BEB)approachimple-
ural selection on the evolution of genes encoding Shiva mented in M8 was only able to identify one positively se-
superfamily toxins (see Methods section for full details lected site in the combined toxin dataset (Table 2 and
of the selection analyses). The one-ratio model, the sim- Additionalfile 1: Table S3). Thus, the site-specific models
plest of the codon-specific models, estimated the non- failed to detect the influence of adaptive selection pres-
synonymous-to-synonymous nucleotide-substitution rate sures in shaping evolution of the Shiva superfamily. In
ratio (ω) to be 0.64, 1.06 and 0.69 for the ω-HXTXs, contrast,themoreadvancedFast,UnconstrainedBayesian
κ-HXTXs, and combined Shiva superfamily dataset, re- AppRoximation (FUBAR) [55,56] implemented in HyPhy
spectively (Additional file 1: Table S1–3). This highly detected a handful of positively selected sites in both the
conservative model can only detect positive selection ω-HXTXsandthecombineddataset(Table1).
when ω, averaged over all sites along the lineages in a Site-specific models for detecting positive selection
phylogenetic tree, is significantly greater than one. As work best when detecting pervasive selection pressures.
lineage-specific models of PAML, such as the one-ratio However, the majority of positively selected sites are
model, often fail to detect positive-Darwinian selection oftensubjectedtotransientorepisodicadaptations.When
thatonlyaffectscertainsitesinproteins,wealsoemployed the majority of lineages evolve under the influence of
Table1Nucleotideandcomplementaryproteinanalysesforωtoxins
Sitea CodeML TreeSAAP Accessible
Codon AminoAcid M2ab M8c Propertyd Magnitudee surfaceareaf
21 E 0.99±0.38 0.87±0.42 - - -
(0.201) (0.257)
40 V 1.41±0.49 1.47±0.37 M ,M,V0,μ 8,8,8,8 42.0
W V
(0.589) (0.842) Partiallyexposed
44 S 1.25±0.37 1.28±0.41 MW,M,V0,μ 8,8,8,8 82.1
V
(0.420) (0.647) Exposed
53 H 1.62±0.63 1.55±0.36 - - 0.0
(0.749) (0.929) Buried
57 G 1.39±0.48 1.45±0.38 - - 57.3
(0.570) (0.825) Exposed
60 T 0.80±0.44 0.71±0.41 - - 49.7
(0.122) (0.154) Exposed
64 N 0.53±0.40 0.51±0.30 - - 100.0
(0.031) (0.036) Exposed
69 T 1.34±0.45 1.38±0.40 - - 59.8
(0.507) (0.751) Exposed
72 R 1.10±0.33 1.01±0.42 - - 0.0
(0.26) (0.374) Buried
aSitesdetectedaspositivelyselectedusingtheintegrativeapproach.
bM2aBayesempiricalBayes(BEB)posteriorprobabilityandpost-meanωindicatedinparentheses.
cM8BayesempiricalBayes(BEB)posteriorprobabilityandpost-meanωindicatedinparentheses.
dAminoacidpropertyunderselection(M :molecularweight;M:molecularvolume;V0:partialspecificvolume;μ:Refractiveindex).
W V
eMagnitudeifselectionontheaminoacidproperty.
fAccessiblesurfaceareaof10–20%correspondstoburiedresidues,40–50%indicatespartiallyexposedaminoacidresidues,and≥50%indicatessolvent
exposedresidues.
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Table2Molecularevolutionofωandκtoxinsfrom [59-64].Sincethesynthesisandsecretionof venompro-
Australianfunnel-webspiders teins is energetically expensive [65-67], mutations that
FUBARa MEMEb PAMLc disrupt the structure/function of proteins are filtered
M8 M2a outofthepopulationbynegativeselectionovertime,fa-
ω ω>1d:3 7 0 0 voring the conservation of catalytic and structurally im-
portant residues. RAVER not only aids in generation of
toxins ω<1e:5 0 0
a rapidly variable toxin molecular surface biochemistry,
0.69 0.73
but it also ensures the conservation of structurally and
κ ω>1a:1 0 0 0 functionally important residues. Accumulation of varia-
toxins ω<1b:0 0 0 tions on the molecular surface of the toxin is advanta-
1.06NS 1.06NS geous as the altered surface chemistry might lead to
ALL ω>1a:3 8 1 0 newtoxinfunctions(neofunctionalisation).
Toderivefurthersupportforthepositivelyselectedsites
toxins ω<1b:7 (0+1) 0
detected by nucleotide analyses, we employed a comple-
0.78 0.83
mentaryprotein-levelapproachimplementedinTreeSAAP
aFast,UnconstrainedBayesianApproximation(FUBAR). (Table1).TreeSAAPidentifiedtwopositivelyselectedsites
bSitesdetectedasexperiencingepisodicdiversifyingselection(0.05
significance)bymixedeffectsmodelevolution(MEME). intheω-HXTXsthatwereincommonwiththesitesiden-
cPositivelyselectedsitesdetectedusingtheBayesempiricalapproach tified by site-model 8 of PAML (Table 1). Evolutionary
implementedinthesitemodelsM8andM2a.Numberofpositivelyselected
sitesdetectedattheposteriorprobability≥0.99and0.95areindicatedin fingerprintanalyses(Figure5A)clearlyrevealedseveralres-
parenthesis.ωcomputedusingM8andM2aarealsopresented. iduesintheω-HXTXsandthecombinedtoxindatasetthat
dNumberofsitesevolvingundertheinfluenceofpervasivediversifying
evolve under the influence of positive selection, while a
selection,detectedbyFUBARat0.9posteriorprobability.
eNumberofsitesevolvingundertheinfluenceofpervasivepurifying majorityofresiduesinthe κ-HXTXsremained under evo-
selection,detectedbyFUBARat0.9posteriorprobability. lutionary constraint (Figure 5A,B). Thus, evolution of the
ω=meandN/dS. ω-HXTXshasbeensignificantlyinfluencedbyshortbursts
NS=notsignificantat0.05comparedtothenullmodel(M7:beta).
of episodic adaptations, while the κ-HXTXs appear to be
negative selection, they mask the signal of positive selec- undernegativeselection.
tion that influences only a small number of lineages. In Phylogeneticanalysisrevealedthattheκ-HXTXsform
such scenarios, the aforementioned analyses may fail to a separate clade to the ω-HXTXs, rendering the Shiva
detecttheinfluenceofpositiveselection.Toaddressthe superfamily non-monophyletic (Figure 6). There are
shortcomings of the aforementioned approaches, we also significant variations within the ω-HXTXs suggest-
employed the advanced Mixed Effects Model Evolution ive of functional diversification (Figure 6). The “hybrid”
(MEME) [57], which uses fixed effects likelihood (FEL) ω/κ-HXTXs exhibit functional characteristics of both
along the sites and random effects likelihood (REL) theω-HXTXsand κ-HXTXs astheyblockCa channels
V
acrossthebranchestodetectepisodicdiversifyingselec- (like the ω-HXTXs) as well as K channels (like the κ-
Ca
tion.MEMEiscapableofidentifyingbothpervasiveand HXTXs). The functional activity of the ω/κ-HXTXs
episodicadaptations.MEMEidentified7and8episodic- combined with their relative phylogenetic placement and
ally diversifying sites in the ω-HXTXs and combined cysteine pattern indicates that they are structurally and
toxin dataset, respectively (Table 2), highlighting the functionally intermediate between the ω- and κ-HXTXs.
vital role of episodic diversifying selection in shaping The evolution of new cysteine residues to create the vici-
theevolutionofthesespidertoxins.Sixoutofeightepi- nal disulfide bond in the κ-HXTXs potentiated toxin
sodically diversifying sites (75%) were located on the activity on K channels, since mutagenesis and analogue
Ca
molecular surface of the toxins (Table 1 and Figure 5B) studiesindicatethatthisvicinaldisulfidebondisthemost
withtheir side chains completely orpartially exposed to criticalpartoftheK pharmacophore[43,45,68].
Ca
solvent, suggesting that they could act as pharmaco-
logical sitesand participateinprey envenomation; these Constraintsonmutationofthematuretoxinsequence
findings are also in agreement with the selection forces It is generally considered that conservation of the cyst-
found on the surface of the SGTx toxin family from the eine scaffold in toxin-gene superfamilies is critical for
venom of the African Baboon spider Scodra grisiepies conserving the toxin’s 3D fold [35]. However, the incred-
[58].RapidAccumulationofVariationsinExposedResi- ible disparity in the amino acid sequence between ω-
dues (RAVER), where the toxin molecular chemistry HXTX-Hv1a and κ-HXTX-Hv1c (Figure 7A) begs the
undergoes hypervariations under the influence of posi- questionofwhetherthisisreflectedinasignificantdiffer-
tiveDarwinianselectionandfocal mutagenesis[59],has enceintheir3Dstructures,despitetheircommoncystine-
been documented in a plethora of venom-components knot scaffold. The 3D structure of both toxins has
from a wide diversity of venomous animal lineages been determined previously using homonuclear NMR
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Figure5Molecularevolutionanalysesofκ-andω-HXTXs.(A)Evolutionaryfingerprintofκ-andω-HXTXs.Estimatesofthedistributionof
synonymous(α)andnon-synonymous(β)substitutionratesinferredfortheκ-HXTXs,ω-HXTXs,andthecombinedShivasuperfamilydataset.The
ellipsesreflectaGaussian-approximatedvarianceineachindividualrateestimate,andcoloredpixelsshowthedensityoftheposteriorsampleof
thedistributionforagivenrate.Thediagonallinerepresentstheidealizedneutralevolutionregime(ω=1),whilepointsaboveandbelowthe
linecorrespondtopositiveselection(ω>1)andnegativeselection(ω<1),respectively.Theωforsitemodel8,alongwiththetotalnumberof
positivelyselectedsitesdetectedbyitsBayesEmpiricalBayes(BEB)approachandthenumberofepisodicallydiversifyingsitesdetectedbythe
mixedeffectsmodelofevolution(MEME),arealsoindicated.(B)MolecularevolutionofShivasuperfamilytoxinsfromAustralianfunnel-web
spiders.3Dhomologymodelsareshownwiththeirmolecularsurfacecoloredaccordingtotheevolutionaryconservationofaminoacids(see
colorkey);thelocationofpositivelyselectedsitesisshowninredinspace-fillmodelsandasredspheresinwireframemodels.Alineplotisalso
providedtohighlighttherelativeaccumulationofdNversusdS,estimatedusingtheM0modelofPAML.NS:Notsignificant.
spectroscopy[18,42]andtheirpharmacophoreselucidated structural differences between the two toxins are the
usingalaninescanningmutagenesis[17,21,43,69]. very different orientations of loops 2 and 3. However,
Figure 7B and C show schematic representations of these structural variations cannot disguise the fact that
the 3D structure of κ-HXTX-Hv1c and ω-HXTX-Hv1a, the two toxins essentially conform to the same 3D scaf-
respectively. The two toxins can be considered to com- fold despite their extraordinary sequence divergence
prisefourinter-cystineloops,whicharelabelled1–4from (16% identity if the cysteine framework is excluded).
N-toC-terminus.Althoughthere is anobvious similarity This ability to maintain a consistent molecular architec-
in the disposition of the three centrally located disulfide ture despite massive variation in the inter-cystine loop
bridgesthatformthecystine-knotmotifineachtoxin,the sequenceshasimportantimplicationsforthemechanism
overall topology of the toxins, as well as the size and bywhich thissuperfamilyofpeptidetoxinshasevolved.
relative orientation of the four inter-cystine loops, ap-
pears quite different. However, the structural overlay in Conclusions
Figure 7D, which was generated automatically by the Spidersandother venomousanimalsrelyontheproduc-
DaliLite structural alignment program [70], reveals that tion of pharmacologically complex venoms for defense,
thetwostructuresareinfactremarkablysimilar. preycapture,andcompetitordeterrence.Themajorcom-
The DaliLite alignment yields a root mean square ponentsofmostspider venomsaredisulfide-richpeptides
deviation of 2.4 Å over the backbone atoms of the 28 that have evolved to target a wide range of receptors and
aligned residues, indicating that the two toxins are in- ion channels in the insect nervous system. The ω-HXTX
deed structural homologs. The three central disulfide andκ-HXTXfamilieswerethefirstpeptidesisolatedfrom
bridges and loop 1 align remarkably well. Loop 4, which Australian funnel-web spiders that were shown to be in-
encompasses the β-hairpin present in both toxins, also secticidal [17]. Analysis of all transcripts encoding these
aligns well except for the four-residue insertion in peptides showed that they are initially expressed as pre-
ω-HXTX-Hv1a (Figure 7A), which increases the size of propeptides that are proteolytically processed to yield a
the hairpin loop at the tip of Loop 4. The major 36–37residuematurepeptidethatcontainsthreedisulfide
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Figure6BayesianphylogenetictreerepresentingthemolecularevolutionaryhistoryoftheShivasuperfamilytoxins.Thetreeshows
thesplitbetweenthethreemaintoxinclasses(ω,κ,andω/κ).ω-Actinopoditoxin-Mb1afromtheEasternmousespiderMissulenabradleyiwas
usedastheoutgroup.Toxinsbelongingtoeachspeciesarehighlightedinthefollowingcolours:H.versuta,red;H.modesta,black;H.venenata,
green;H.infensa,magenta;A.robustus,paleblue;H.formidabilis,darkblue;ω-actinopoditoxin-Mb1afromM.bradleyi,orange.*denotesaspecies
notsequencedaspartofthisstudy;thesequencewasdownloadedfromUniProtunderaccessionnumberP83588.
bridgesthatformanICKmotifplusanon-canonical vici- pharmacologicalsites.Thesetoxinsmaythereforebegood
naldisulfidebondintheκ-HXTXs. candidates for in vitro evolution studies designed to pro-
The extreme diversity of primary structure within the duce modified peptides with desired therapeutic [14] or
Shiva toxin superfamily suggests that there have been agrochemical[11]properties.Mostimportantly,thisstudy
few evolutionary restraints on sequence diversification reinforces the idea that the remarkable chemical and
outside of the disulfide bridges that direct the 3D fold of pharmacological complexity of spider venoms may bede-
these peptides. The ω-HXTXs, in particular, seem to rivedfromarelativelysmallnumberofancestralgenes.
have evolved under the influence of positive Darwinian
selection in an episodic fashion, whereas the κ-HXTXs Methods
appear to be constrained by negative selection pressures. Identificationofω-HXTX-1homologsviarapid
Functional assessments of these toxins should shed fur- amplificationofcDNAends
ther light on why they have adopted quite contrasting Venom-gland cDNA libraries were prepared from indi-
molecular evolutionary regimes. ω-HXTXs were also vidual specimens of the following species of Australian
found to have adopted RAVER, where a large number of funnel-web spider (Arthropoda: Chelicerata: Arachnida:
the episodically diversifying sites are concentrated on Araneae: Opisthothelae: Mygalomorphae: Hexathelidae):
themolecularsurface,facilitatingthegeneration ofnovel Hadronyche infensa, H. versuta, H. venenata, and Atrax