Toxins

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Submit to this Journal Review for this Journal Edit a Special Issue ► ▼ Article Menu Article Menu Subscribe SciFeed Recommended Articles Related Info Links PubMed/Medline Google Scholar More by Authors Links on DOAJ Sunagar, K. Jackson, T. N. W. Undheim, E. A. B. Ali, S. A. Antunes, A. Fry, B. G. on Google Scholar Sunagar, K. Jackson, T. N. W. Undheim, E. A. B. Ali, S. A. Antunes, A. Fry, B. G. on PubMed Sunagar, K. Jackson, T. N. W. Undheim, E. A. B. Ali, S. A. Antunes, A. Fry, B. G. /ajax/scifeed/subscribe Article Views Citations - Table of Contents Altmetric share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up ... Endorse textsms ... Comment Need Help? Support

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Get Information clear JSmol Viewer clear first_page settings Order Article Reprints Font Type: Arial Georgia Verdana Font Size: Aa Aa Aa Line Spacing:    Column Width:    Background: Open AccessArticle Three-Fingered RAVERs: Rapid Accumulation of Variations in Exposed Residues of Snake Venom Toxins by

Kartik Sunagar

Kartik Sunagar Scilit Preprints.org Google Scholar 1,2,

Timothy N. W. Jackson

Timothy N. W. Jackson Scilit Preprints.org Google Scholar 3,4,

Eivind A. B. Undheim

Eivind A. B. Undheim Scilit Preprints.org Google Scholar 3,4,

Syed. A. Ali

Syed. A. Ali Scilit Preprints.org Google Scholar 3,4,5,

Agostinho Antunes

Agostinho Antunes Scilit Preprints.org Google Scholar 1,2 ORCID

and

Bryan G. Fry

Bryan G. Fry Scilit Preprints.org Google Scholar 3,4,* ORCID

1 CIMAR/CIIMAR, Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Rua dos Bragas, 177, Porto 4050-123, Portugal 2 Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, Porto 4169-007, Portugal 3 Venom Evolution Lab, School of Biological Sciences, The University of Queensland, St. Lucia, Queensland 4072, Australia 4 Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4072, Australia 5 HEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi-75270, Pakistan * Author to whom correspondence should be addressed. Toxins 2013, 5(11), 2172-2208; https://doi.org/10.3390/toxins5112172 Received: 2 October 2013 / Revised: 8 November 2013 / Accepted: 11 November 2013 / Published: 18 November 2013 (This article belongs to the Collection Evolution of Venom Systems) Download Download PDF Download PDF with Cover Download XML Download Epub Download Supplementary Material Browse Figures Versions Notes

Abstract: Three-finger toxins (3FTx) represent one of the most abundantly secreted and potently toxic components of colubrid (Colubridae), elapid (Elapidae) and psammophid (Psammophiinae subfamily of the Lamprophidae) snake venom arsenal. Despite their conserved structural similarity, they perform a diversity of biological functions. Although they are theorised to undergo adaptive evolution, the underlying diversification mechanisms remain elusive. Here, we report the molecular evolution of different 3FTx functional forms and show that positively selected point mutations have driven the rapid evolution and diversification of 3FTx. These diversification events not only correlate with the evolution of advanced venom delivery systems (VDS) in Caenophidia, but in particular the explosive diversification of the clade subsequent to the evolution of a high pressure, hollow-fanged VDS in elapids, highlighting the significant role of these toxins in the evolution of advanced snakes. We show that Type I, II and III α-neurotoxins have evolved with extreme rapidity under the influence of positive selection. We also show that novel Oxyuranus/Pseudonaja Type II forms lacking the apotypic loop-2 stabilising cysteine doublet characteristic of Type II forms are not phylogenetically basal in relation to other Type IIs as previously thought, but are the result of secondary loss of these apotypic cysteines on at least three separate occasions. Not all 3FTxs have evolved rapidly: κ-neurotoxins, which form non-covalently associated heterodimers, have experienced a relatively weaker influence of diversifying selection; while cytotoxic 3FTx, with their functional sites, dispersed over 40% of the molecular surface, have been extremely constrained by negative selection. We show that the a previous theory of 3FTx molecular evolution (termed ASSET) is evolutionarily implausible and cannot account for the considerable variation observed in very short segments of 3FTx. Instead, we propose a theory of Rapid Accumulation of Variations in Exposed Residues (RAVER) to illustrate the significance of point mutations, guided by focal mutagenesis and positive selection in the evolution and diversification of 3FTx. Keywords: positive selection; venom evolution; three-finger toxins; RAVER; focal mutagenesis 1. IntroductionVenoms are key evolutionary innovations in the Kingdom Animalia and are complex concoctions of biologically active proteins (from polypeptide globular enzymes to small peptides), salts, and organic molecules such as polyamines, amino acids and neurotransmitters [1]. Venom components originate via toxin recruitment events during which ordinary protein-encoding genes, typically those involved in key regulatory processes (such as hemostasis or neurotransmission) are duplicated, and the new copies are selectively expressed in the venom gland [1,2,3,4,5,6,7,8,9,10,11,12,13]. These novel paralogs can further duplicate and give rise to multigene families, following the “birth and death” mode of evolution, where the rapid evolution of these families results in extensive neofunctionalization of some copies, while the other non-functional forms are lost through degradation or get transformed into pseudogenes [14]. Research has shown that despite the extraordinary diversity of animal toxins, most belong to a limited number of enzymatic (e.g., phospholipases, serine proteases, metalloproteinases) and non-enzymatic (e.g., three-finger toxins, natriuretic peptides, Kunitz peptides, lectins) protein superfamilies which have been convergently recruited in various organisms to perform similar functions [1,3].Three-finger toxins (3FTx) are one of the most abundantly secreted non-enzymatic components of elapid (Elapidae), colubrid (Colubridae) and psammophiide (Psammophiinae subfamily withing the Lamprophidae) snake venom. They are characterised by a broad diversity of functional forms (Table 1). In the past, 3FTx were considered to be exclusive to elapid snake venoms [6]. The discovery of α-colubritoxin, however, revealed this potent toxin type to be widespread in “non-front-fanged” (NFF) Caenophidia snake lineages [15]. Subsequent studies revealed the broad taxonomic distribution of this toxin type [16,17,18,19,20,21,22], which appears to have been recruited into the snake venom arsenal near the base of the snake tree [9]. 3FTx are characterised by the presence of three β-loops that extend from the toxin’s small, hydrophobic core, giving them the “three-fingered” appearance and their name [23]. The plesiotypic (ancestral character state of a molecular scaffold) 3FTx form, such as that found in NFF advanced snakes, has ten cysteines in a distinctive pattern, reflective of its molecular origin from the recruitment of a LYNX/SLUR nicotinic receptor binding neuromodulation peptide [3,6,15,17]. All described 3FTx types from Henophidia, NFF and viperid (Viperidae) snakes contain these ten cysteines, and all of these forms that have been functionally investigated are α-neurotoxic, with a potency much greater against birds and/or reptiles than mammals [6,9,17,19,20,21,24]. This taxon specificity in action led to the plesiotypic α-neurotoxins being mistakenly referred to as “weak neurotoxins” (c.f. [25]). Similarly, this taxon-specific toxicity led to misinterpretation of prey-handling behaviour in an experiment investigating the role of venom in the feeding ecology of NFF snakes [26]. Table

Table 1. Bioactivities of 3FTx types with characterised toxicities. Table 1. Bioactivities of 3FTx types with characterised toxicities. Functional ClassMode of ActionBasal-type α-neurotoxinsAntagonists of α1 nicotinic acetylcholine receptors, with a 100-fold greater potency to avians/reptiles than mammals. Produces flaccid paralysis [21]Type I α-neurotoxinsAntagonists of α1 nicotinic acetylcholine receptors. Produces flaccid paralysis [27]Type II α-neurotoxinsAntagonists of α1 and α7 nicotinic acetylcholine receptors. Produces flaccid paralysis [27]Type III α-neurotoxinsAntagonists of α1 nicotinic acetylcholine receptors. Produces flaccid paralysis [28] κ-neurotoxinsAntagonists of α3β2 neuronal nicotinic acetylcholine receptor subtype. Produces flaccid paralysis [29]Adrenergic/Muscarinic neurotoxinsAntagonists of a wide variety of adrenergic and muscarinic subtypes with extreme specificity for receptor subtypes [30,31,32,33,34,35,36,37,38,39,40,41]Type B Muscarinic toxinsAntagonists of M2 muscarinic acetylcholine receptors [42]ASIC channel blockersActs as a reversible gating modifier toxin by antagonistically binding to closed/inactivated ASIC1a-ASIC2a (ACCN2-ACCN1) channels in central neurons and ASIC1b-containing channels in nociceptors [43]Calcium channel blockersAntagonists of L-type calcium channels, thus inhibiting the transmission of the action potential [44]Acetylcholinesterase inhibitorsInhibit acetylcholinesterase through competitive binding [45]Platelet inhibitorsCompetitively bind to platelet GPIIb/IIIa receptor utilising the RGD functional motif, thus blocking platelet aggregation [46]CytotoxinsCell-damaging activity mediated by hydrophobic-patch on molecular surface that interacts non-specifically with hydrophobic aspects of the cell phospholipid bilayer [47]SynergisticAlone are non-toxic but form complexes with α-neurotoxins to dramatically enhance neurotoxicity [48] Toxins 05 02172 g001 1024

Figure 1. Bayesian molecular phylogeny of representative three-finger toxins. Uniprot [49] accession numbers are given for each. Cysteine framework variation is displayed, with ancestral cysteines in black and newly evolved cysteines in red. Figure 1. Bayesian molecular phylogeny of representative three-finger toxins. Uniprot [49] accession numbers are given for each. Cysteine framework variation is displayed, with ancestral cysteines in black and newly evolved cysteines in red. Toxins 05 02172 g001

Subsequent to elapid snakes evolving a high pressure, syringe-like delivery system including venom gland compressor musculature and hollow front fangs, apotypic (derived character state of a molecular scaffold) forms of 3FTx emerged, characterised by a loss of plesiotypic cysteines 2 and 3 (Figure 1), a change that probably resulted in the dramatic potentiation of α-neurotoxicity through the uncoupling of loop-1, with these apotypic forms becoming much more potent upon mammalian receptors than the more constrained plesiotypic forms [6]. This increased toxicity likely enhanced the role of these proteins in prey capture and resulted in a high level expression of α-neurotoxins (α-ntx) lacking the second and third plesiotypic cysteines (Type I (aka: short-chain) and Type II (aka: long-chain) α-ntx) in the venom glands of these snakes. The increased level of expression was accompanied by punctuated molecular evolution, resulting in the emergence of a myriad of structurally and functionally novel forms [6]. Structurally novel forms included α-ntx with newly evolved cysteines that stabilised the second loop (Type II α-ntx). Venoms of snakes from the Oxyuranus and Pseudonaja clade are unusual in containing significant amounts of 3FTx that display all the features of Type II α-ntx (including lacking the 2nd and 3rd plesiotypic cysteines) but lack the apotypic loop-2 stabilising cysteine pair characteristic of this type [50]. These toxins would thus be expected to be phylogenetically basal to the Type II α-ntx which contain the apotypic loop-2 stabilising cysteine pair. Another derived 3FTx structural variation is represented by the κ-neurotoxins (aka: κ-bungarotoxins), which form non-covalently associated heterodimers. A number of novel functions also emerged ([6]; Table 1; Figure 1), such as κ-ntx specifically targeting the neuronal nicotinic receptors. The most extreme neofunctionalisation is represented by the cytotoxins, which have deviated from the highly focused ion channel targeting of the α-ntx [51]. Instead, they exhibit a number of novel biological activities, including lysis of various types of cells (including erythrocytes and epithelial cells), enzyme inhibition (protein kinase C: [52]; Na+/K+ ATPase: [53]), depolarization and contraction of muscle cells and prevention of platelet aggregation. All structurally and functionally apotypic 3FTxs lack plesiotypic cysteines 2 and 3 (Figure 1), and are expressed in the venoms of elapid snakes in much higher levels than the α-ntxs that contain all ten plesiotypic cysteines.Although it has been hypothesised and demonstrated through (now obsolete) selection assessments that snake venom three-finger toxins have evolved under the influence of positive Darwinian selection [6,54,55], the underlying mechanism of evolution driving the diversification of functional forms remains unclear. The molecular evolution of several 3FTx forms, such as κ-ntx, plesiotypic 3FTxs from Henophidia, NFF and elapid snakes and Type III α-neurotoxins from Australian elapids, remain unstudied to date. Molecular mechanisms underlying the evolution of the 3FTx gene in viperid snakes, which have independently evolved a sophisticated high pressure, hollow-fanged venom delivery system (VDS), also remain elusive. Moreover, interpretations regarding the evolution of certain 3FTx types, such as the suggestion that cytotoxins evolve under the influence of positive selection (with an ω value of 19.5) [55], have been questionable. Recently, the molecular evolution of 3FTx homologues in Atractaspis sp. was evaluated and the influence of positive Darwinian selection on genes encoding them was highlighted [56].As the general organization of the 3FTx gene is highly conserved and ordered, Accelerated Segment Switch in Exons to alter Targeting (ASSET) has been postulated as the mechanism driving the molecular evolution of 3FTx [16,57]. This theory suggests that during the evolution of the 3FTx gene, segments in exonic regions have been exchanged with distinctly different ones and the resultant “switching” of segments has generated the observed sequence variation and the functional diversity of 3FTx. The authors further speculated that point mutations alone could not account for the diversity of 3FTx functional forms and that they could only be helpful in fine tuning receptor binding capabilities, which originally arise through ASSET [16,57]. However, this study attempted to classify regions in 3FTx based on simplistic “degree of identity” comparisons. Crucially, such analysis does not take into account the fact that proteins adopt regionally differential rates of evolution [58,59,60]. It has been previously demonstrated in other toxin types that structurally important residues are constrained by negative selection, while regions responsible for biological function and/or those forming the molecular surface accumulate variations under an arms race scenario [58,59,61]. This not only facilitates functional diversification, but also increases the number of active residues on the surface of venom components that can non-specifically interact with novel receptors in prey and induce a plethora of pharmacological effects.In the present study, we test the following hypotheses: (i) that 3FTx with specific sites of action are involved in a coevolutionary arms race with receptors of prey animals and thus are evolving under the influence of positive selection; (ii) that cytotoxic 3FTx, which interact non-specifically with cell membranes, do not experience an arms race and evolve under the constraints of negative selection pressure; (iii) that, in venoms in which 3FTx are a major component (Elapidae, Colubridae and Psammophiinae), 3FTx are rapidly evolving under positive selection (with the exception of cytotoxic 3FTx in the venom of elapid snakes); (iv) that in venoms in which 3FTx are a minor component (Viperidae), 3FTx evolve under a neutral selection regime and do not experience positive selection; and (v) the evolution of the advanced venom delivery system in elapid snakes led to an increase in diversifying pressure on 3FTx, resulting in the diversity of functional forms present in the venoms of snakes from this family. In order to test these hypotheses and provide further insight into the evolution and diversification of this toxin superfamily, we reconstructed the complex molecular evolutionary history of 3FTx. In particular, this study examined: the relative rate of evolution of the plesiotypic α-ntx (i) before and after the evolution of the advanced venom delivery apparatus in Caenophidia (advanced snakes), in order to test whether the evolution of a sophisticated VDS influenced the regime of 3FTx evolution; (ii) before and after the evolution of the sophisticated, high pressure and hollow-fanged venom delivery system in elapids and viperids to test whether the refining of VDS influenced selection pressures on 3FTx genes; (iii) the relative rate of 3FTx evolution subsequent to the loss of two plesiotypic cysteines and resultant potentiation of α-neurotoxicity; (iv) the relative rate of evolution subsequent to the apotyposis (or derivation) of non-covalently associated dimeric forms, in order to find out whether these new structural constraints affected natural selection pressures on this gene; and finally; (v) the relative rate of evolution subsequent to the apotyposis of cytotoxins was examined to understand whether the novel activity recruited by cytotoxins affected their rate of evolution. 2. ResultsBayesian and maximum-likelihood molecular phylogenetic analyses retrieved phylogenetic trees with similar topologies (Figure 1; Supplementary Figure 1), with different toxin sequences forming distinct phylogenetic clades as shown previously [3,6,17]. The Bayesian tree (convergence diagnostics: Average standard deviation of split frequencies: 0.009; PSRF = 1.00) was built using amino acid sequences, since some 3FTxs are only known from their amino acid sequences, while the maximum-likelihood tree (1,000 bootstrap replicates; GTR + I + G) was built using all the nucleotide sequences analysed in this study (n = 457). The overall topology of these phylogenetic trees was largely in concordance with the previously published 3FTx phylogeny [3,6,17].A particularly notable finding was that the novel Type II α-ntx sequences from Oxyuranus and Pseudonaja, which lack the Type II α-ntx characteristic cysteine doublet (−2C) between plesiotypic cysteines 5 and 6, were not phylogenetically basal to the other Type II, nor were they monophyletic, but rather were nested within the regular Type II forms (+2C) (Figure 2). This suggests that the apotypic cysteine doublet characteristic of Type II α-ntx was secondarily lost on at least three occasions. Toxins 05 02172 g002 1024

Figure 2. Bayesian molecular phylogeny and structural and functional evolution of +2C/−2C Type II (long-chain) α-neurotoxins. Pseudonaja/Oxyuranus −2C and +2C sequences are coloured purple and green, respectively. Sequences presented (uniprot): (1) R4FIT5 Pseudonaja modesta, (2) R4FIU6 Pseudonaja modesta, (3) A8HDK6 Pseudonaja textilis, (4) R4FK68 Pseudonaja modesta, (5) A8HDK8 Oxyuranus microlepidotus, (6) A7X4Q3 Oxyuranus microlepidotus, (7) A8HDK7 Oxyuranus microlepidotus, (8) A7X4R0 Oxyuranus microlepidotus, (9) A8HDK9 Oxyuranus scutellatus, (10) Q9W7J5 Pseudonaja textilis, (11) R4FIT0 Pseudonaja modesta, (12) R4G7K3 Pseudonaja modesta, (13) R4G321 Pseudonaja modesta, (14) R4G2J4 Pseudonaja modesta, (15) R4G319 Pseudonaja modesta and (16) R4FK75 Pseudonaja modesta. Figure 2. Bayesian molecular phylogeny and structural and functional evolution of +2C/−2C Type II (long-chain) α-neurotoxins. Pseudonaja/Oxyuranus −2C and +2C sequences are coloured purple and green, respectively. Sequences presented (uniprot): (1) R4FIT5 Pseudonaja modesta, (2) R4FIU6 Pseudonaja modesta, (3) A8HDK6 Pseudonaja textilis, (4) R4FK68 Pseudonaja modesta, (5) A8HDK8 Oxyuranus microlepidotus, (6) A7X4Q3 Oxyuranus microlepidotus, (7) A8HDK7 Oxyuranus microlepidotus, (8) A7X4R0 Oxyuranus microlepidotus, (9) A8HDK9 Oxyuranus scutellatus, (10) Q9W7J5 Pseudonaja textilis, (11) R4FIT0 Pseudonaja modesta, (12) R4G7K3 Pseudonaja modesta, (13) R4G321 Pseudonaja modesta, (14) R4G2J4 Pseudonaja modesta, (15) R4G319 Pseudonaja modesta and (16) R4FK75 Pseudonaja modesta. Toxins 05 02172 g002

Our analysis of the venom gland transcriptome of the unique viperid species Azemiops feae recovered a 3FTx transcript that was found in the same clade as the representative sequences from the pit-viper Sistrurus catenatus (Figure 1; Supplementary Figure 1). This is the first non-Sistrurus 3FTx to be recovered from a viperid snake. Based on an unresolved neighbour joining tree, viperid 3FTx were previously reported as polyphyletic, with some sequences nested within elapid 3FTx clade, and others in the “non-front-fanged” advanced snake 3FTx clade [24]. However, both Bayesian and maximum-likelihood phylogenetic analyses in this study place all viperid sequences in a monophyletic clade outside elapid 3FTx clade with strong node support (Figure 1; Supplementary Figure 1). This is consistent with the viperid snakes having diverged from the remaining advanced snakes nearly 54 million years ago [62]. viperid 3FTx homologs retrieved to date indicate that apotypic structural and functional forms, such as Type I, II and III α-ntxs, κ-ntxs and cytotoxins, are the result of apotyposis in elapid snakes after this split (Figure 1; Supplementary Figure 1).The one-ratio model (ORM), the simplest of the lineage-specific models, computed a wide range of ω (dN/dS) values for various types of 3FTx (Supplementary Tables 1–10). This highly conservative model can only detect positive selection when the ω ratio, averaged over all sites along the lineages in a phylogenetic tree, is significantly greater than one. Despite this, the ω value for most three-finger toxins was significantly greater than 1, indicating the strong influence of positive selection in shaping the evolution of 3FTx: plesiotypic α-ntx from viperids, NFF and elapids: 1.79, 1.29 and 1.30, respectively; Type I, Type II and Type III α-ntx: 1.92, 2.01 and 2.59, respectively; and κ-ntx: 1.64. Oxyuranus/Pseudonaja Type II α-ntx with (+2C) and without the cys doublet (−2C) were significantly different, with ω values of 0.97 and 2.69, respectively. In contrast to all other 3FTx types, ORM estimated an ω value of 0.32 for cytotoxins, indicating an unprecedented lack of variation in this unique three-finger toxin.The segregation of seromucous mixed maxillary glands into discrete protein and mucus glands at the base of the advanced snake tree and the subsequent evolution of a high pressure, hollow-fanged venom delivery apparatus independently in Atractaspis/Homoroselaps, elapids and viperids were major evolutionary advances in snake-feeding ecology [7,17]. In order to assess the effect of apomorphic (derived state of a morphological character) venom delivery systems on the evolution of 3FTx, we employed the three-ratio model (3RM) and the branch-site test A (BST) on the plesiotypic α-ntx from the advanced snakes (NFF, viperids and elapids). While 3RM—which assesses selection pressure only across lineages—estimated ω of 1.13, 1.37 and 2.21 for the plesiotypic α-ntx from NFF, elapids and viperids, respectively; BST—which assess selection pressures across the sites and along the lineages in a tree—estimated ω of 2.16 (22% sites), 4.09 (23% sites) and 6.54 (31% sites), respectively (Table 2). NFF and elapid comparisons were insignificant (p > 0.05) for 3RM, while all other comparisons were significant (significant at 0.001 even after Bonferroni corrections). 3RM and BST indicated a greater influence of positive selection on the plesiotypic α-ntxs in elapid and viperid snakes, which have independently evolved sophisticated high pressure and hollow-fanged venom delivery systems. Table

Table 2. Lineage-specific analyses of plesiotypic Caenophidia three-finger toxins (3FTxs). Table 2. Lineage-specific analyses of plesiotypic Caenophidia three-finger toxins (3FTxs). Modelω aLikelihood (ι)Prop. of Sites with ω > 1 bSignificance cPlesiotypic α-neurotoxins from ‘non-front-fanged’ advanced snakesThree-ratio model1.13−5982.232579-P > 0.05 NSBranch-site model A2.16−5767.71045422.1%* P 1 (positive selection) and M7 (Beta) versus M8 (Beta and ω), and models that mirror the evolutionary constraints of M1 and M2 but assume that ω values are drawn from a beta distribution [125]. Only if the alternative models (M3, M2a and M8: allow sites with ω > 1) show a better fit in Likelihood Ratio Test (LRT) relative to their null models (M0, M1a and M7: do not allow sites ω > 1), are their results considered significant. It should be noted that although we have reported the results of all site-specific models (M2a, M3 and M8), only the results of M8, the most accurate model among these, were considered for downstream analyses (Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, and Figure 8; Table 4 and Table 5). LRT is estimated as twice the difference in maximum likelihood values between nested models and compared with the χ2 distribution with the appropriate degree of freedom—the difference in the number of parameters between the two models. The Bayes empirical Bayes (BEB) approach [126] was used to identify amino acids under positive selection by calculating the posterior probabilities that a particular amino acid belongs to a given selection class (neutral, conserved or highly variable). Sites with greater posterior probability (PP ≥ 95%) of belonging to the “ω > 1 class” were inferred to be positively selected.FUBAR [127] implemented in HyPhy [128] was employed to detect codon sites evolving under the influence of pervasive diversifying and purifying selection pressures. The Mixed Effects Model of Evolution (MEME) [129] was also employed to efficiently detect sites that experience diversifying selection for a short period in the evolutionary timescale. Non-synonymous mutations that introduce extremely variant amino acids are more likely to influence the structure-function, and hence the fitness of the organism, relative to mutations that introduce amino acids with similar side-chains as the ancestral residues. Further support for the results of the nucleotide-level selection analyses was obtained and the radicalness of mutations were assessed using a complementary protein-level approach implemented in TreeSAAP [130].Direct comparison of ω values computed from different datasets can be misleading, as they can have different proportions of sites under selection. Hence, we assessed the selection pressures shaping different 3FTx clades by employing clade model analyses implemented in Codeml and simultaneously estimated ω values [131]. The significance of the analysis was tested by comparing the likelihood of this model with that of model M1a. To clearly depict the proportion of sites under different regimes of selection, an evolutionary fingerprint analysis was carried out using the evolutionary selection distance (ESD) algorithm implemented in datamonkey [132]. 5.7. Structural AnalysesTo depict the natural selection pressures influencing the evolution of various three-finger toxins, we mapped the sites under positive selection on the homology models created using the Phyre 2 webserver [133]. Pymol 1.3 [134] was used to visualise and generate the images of homology models. The Consurf webserver [135] was used for mapping the evolutionary selection pressures on the three-dimensional homology models. GETAREA [136] was used to calculate the Accessible Surface Area (ASA) or the solvent exposure of amino-acid side chains. It uses the atom co-ordinates of the PDB file and indicates if a residue is buried or exposed to the surrounding medium by comparing the ratio between side chain ASA and the “random coil” values per residue. An amino acid is considered to be buried if it has an ASA less than 20% and exposed if the ASA is more than or equal to 50%. When ASA ratio lies between 40% and 50%, it is highly likely that the residues have their side chains exposed to the surrounding medium. AcknowledgementsKS was funded by the PhD grant (SFRH/BD/61959/2009) from F.C.T (Fundação para a Ciência e a Tecnologia). TNWJ was funded by a PhD scholarship from the University of Queensland. AA was funded by the European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Programme and national funds through F.C.T under the projects PEst-C/MAR/LA0015/2013 and PTDC/AAC-AMB/121301/2010 (FCOMP-01-0124-FEDER-019490), and BGF was funded by the Australian Research Council (ARC) and the University of Queensland. SAA was the recipient of a postdoctoral fellowship (PDRF Phase II Batch-V) from Higher Education Commission (HEC Islamabad) Pakistan. 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Sunagar, K.; Jackson, T.N.W.; Undheim, E.A.B.; Ali, S.A.; Antunes, A.; Fry, B.G. Three-Fingered RAVERs: Rapid Accumulation of Variations in Exposed Residues of Snake Venom Toxins. Toxins 2013, 5, 2172-2208. https://doi.org/10.3390/toxins5112172

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Sunagar K, Jackson TNW, Undheim EAB, Ali SA, Antunes A, Fry BG. Three-Fingered RAVERs: Rapid Accumulation of Variations in Exposed Residues of Snake Venom Toxins. Toxins. 2013; 5(11):2172-2208. https://doi.org/10.3390/toxins5112172

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Sunagar, Kartik, Timothy N. W. Jackson, Eivind A. B. Undheim, Syed. A. Ali, Agostinho Antunes, and Bryan G. Fry. 2013. "Three-Fingered RAVERs: Rapid Accumulation of Variations in Exposed Residues of Snake Venom Toxins" Toxins 5, no. 11: 2172-2208. https://doi.org/10.3390/toxins5112172

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