Three-Fingered RAVERs: Rapid Accumulation of Variations in Exposed Residues of Snake Venom Toxins

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.

Melt With This Kiss: Paralyzing and Liquefying Venom of The Assassin Bug Pristhesancus plagipennis (Hemiptera: Reduviidae)

“…CUB domains also occur as one of several domains in some insect and crustacean S1 proteases, including the bee venom protease Api m 7 (48 -50), and 19 of the 67 S1 proteases detected in P. plagipennis venom. In contrast to this pattern, we observed that two of the most abundant proteins (CUB domain proteins 1 and 2) in P. plagipennis venom consist of a solitary CUB domain; three further CUB domain proteins (3)(4)(5) were detected from HPLC fractions and 1D SDS-polyacrylamide gels. Each CUB domain protein contains 129 -142 residues and is stabilized by two conserved disulfide bonds (Fig.…”

“…Typically, venoms are composed of multiple toxins, including peptides, enzymes, and small molecules, such as polyamines, that bind to and affect the function of multiple molecular targets in the injected animal. Because of their key role governing life-or-death interactions between animals, venom toxins are subject to selection pressures that have resulted in unique evolutionary patterns such as massive duplication and accelerated evolution of toxin-encoding genes (2)(3)(4)(5). In addition, the properties that ensure that toxins confer a fitness advantage to the animals that produce them, including high stability and potency, make them well suited for use as insecticides, therapeutics, and pharmacological tools (6 -11).…”

Deep venomics of the Pseudonaja genus reveals inter- and intra-specific variation

“…However, three sequences displayed nine cysteine residues, including LNTX-2 from P. textilis with a truncated (Table S2), exemplifying the rapid evolution of these toxins [31]. Further, several positively selected sites (ranged between 4-32) were identified in these toxins types (Table S2).…”

“…Members belonging to this toxin superfamily are typically structured with three distinct β-stranded loops extending from a disulfide rich hydrophobic core [31]. α-neurotoxic 3FTxs may be of Types I and III (short-chain) composed of 60-62 residues with 4 disulfides or Type II (long-chain) α-neurotoxin 3FTx composed of 66-74 residues and 5 disulfide bonds.…”

Evolution and diversification of the cnidarian venom system

“…It has been suggested that most venom proteins that are involved in predation evolve through rapid accumulation of variation in the exposed residues (RAVER), where the molecular surface of the toxin accumulates bulk of the variations under the significant influence of positive Darwinian selection, while preserving the core residues involved in stability and/or catalytic activity [184]. As synthesis and secretion of proteins is an energetically expensive process, over time, mutations that lead to the loss of stable structure and function are filtered out of the population by purifying selection.…”