Unique scorpion toxin with a putative ancestral fold provides insight into evolution of the inhibitor cystine knot motif

The three-disulfide inhibitor cystine knot (ICK) motif is a fold common to venom peptides from spiders, scorpions, and aquatic cone snails. Over a decade ago it was proposed that the ICK motif is an elaboration of an ancestral two-disulfide fold coined the disulfide-directed β-hairpin (DDH). Here we report the isolation, characterization, and structure of a novel toxin [U1-liotoxin-Lw1a (U1-LITX-Lw1a)] from the venom of the scorpion Liocheles waigiensis that is the first example of a native peptide that adopts the DDH fold. U1-LITX-Lw1a not only represents the discovery of a missing link in venom protein evolution, it is the first member of a fourth structural fold to be adopted by scorpion-venom peptides. Additionally, we show that U1-LITX-Lw1a has potent insecticidal activity across a broad range of insect pest species, thereby providing a unique structural scaffold for bioinsecticide development

Molecular Evolution, Functional Variation, and Proposed Nomenclature of the Gene Family That Includes Sphingomyelinase D in Sicariid Spider Venoms

The venom enzyme sphingomyelinase D (SMase D) in the spider family Sicariidae (brown or fiddleback spiders [Loxosceles] and six-eyed sand spiders [Sicarius]) causes dermonecrosis in mammals. SMase D is in a gene family with multiple venom-expressed members that vary in functional specificity. We analyze molecular evolution of this family and variation in SMase D activity among crude venoms using a data set that represents the phylogenetic breadth of Loxosceles and Sicarius. We isolated a total of 190 nonredundant nucleotide sequences encoding 168 nonredundant amino acid sequences of SMase D homologs from 21 species. Bayesian phylogenies support two major clades that we name α and β, within which we define seven and three subclades, respectively. Sequences in the α clade are exclusively from New World Loxosceles and Loxosceles rufescens and include published genes for which expression products have SMase D and dermonecrotic activity. The β clade includes paralogs from New World Loxosceles that have no, or reduced, SMase D and no dermonecrotic activity and also paralogs from Sicarius and African Loxosceles of unknown activity. Gene duplications are frequent, consistent with a birth-and-death model, and there is evidence of purifying selection with episodic positive directional selection. Despite having venom-expressed SMase D homologs, venoms from New World Sicarius have reduced, or no, detectable SMase D activity, and Loxosceles in the Southern African spinulosa group have low SMase D activity. Sequence conservation mapping shows >98% conservation of proposed catalytic residues of the active site and around a plug motif at the opposite end of the TIM barrel, but α and β clades differ in conservation of key residues surrounding the apparent substrate binding pocket. Based on these combined results, we propose an inclusive nomenclature for the gene family, renaming it SicTox, and discuss emerging patterns of functional diversification

Palmitoylation influences the function and pharmacology of sodium channels

Palmitoylation is a common lipid modification known to regulate the functional properties of various proteins and is a vital step in the biosynthesis of voltage-activated sodium (Nav) channels. We discovered a mutation in an intracellular loop of rNav1.2a (G1079C), which results in a higher apparent affinity for externally applied PaurTx3 and ProTx-II, two voltage sensor toxins isolated from tarantula venom. To explore whether palmitoylation of the introduced cysteine underlies this observation, we compared channel susceptibility to a range of animal toxins in the absence and presence of 2-Br-palmitate, a palmitate analog that prevents palmitate incorporation into proteins, and found that palmitoylation contributes to the increased affinity of PaurTx3 and ProTx-II for G1079C. Further investigations with 2-Br-palmitate revealed that palmitoylation can regulate the gating and pharmacology of wild-type (wt) rNav1.2a. To identify rNav1.2a palmitoylation sites contributing to these phenomena, we substituted three endogenous cysteines predicted to be palmitoylated and found that the gating behavior of this triple cysteine mutant is similar to wt rNav1.2a treated with 2-Br-palmitate. As with chemically depalmitoylated rNav1.2a channels, this mutant also exhibits an increased susceptibility for PaurTx3. Additional mutagenesis experiments showed that palmitoylation of one cysteine in particular (C1182) primarily influences PaurTx3 sensitivity and may enhance the inactivation process of wt rNav1.2a. Overall, our results demonstrate that lipid modifications are capable of altering the gating and pharmacological properties of rNav1.2a

Novel Venom Proteins Produced by Differential Domain-Expression Strategies in Beaded Lizards and Gila Monsters (genus Heloderma)

The origin and evolution of venom proteins in helodermatid lizards were investigated by multidisciplinary techniques. Our analyses elucidated novel toxin types resultant from three unique domain-expression processes: 1) The first full-length sequences of lethal toxin isoforms (helofensins) revealed this toxin type to be constructed by an ancestral monodomain, monoproduct gene (beta-defensin) that underwent three tandem domain duplications to encode a tetradomain, monoproduct with a possible novel protein fold; 2) an ancestral monodomain gene (encoding a natriuretic peptide) was medially extended to become a pentadomain, pentaproduct through the additional encoding of four tandemly repeated proline-rich peptides (helokinestatins), with the five discrete peptides liberated from each other by posttranslational proteolysis; and 3) an ancestral multidomain, multiproduct gene belonging to the vasoactive intestinal peptide (VIP)/ glucagon family being mutated to encode for a monodomain, monoproduct (exendins) followed by duplication and diversification into two variant classes (exendins 1 and 2 and exendins 3 and 4). Bioactivity characterization of exendin and helokinestatin elucidated variable cardioactivity between isoforms within each class. These results highlight the importance of utilizing evolutionary-based search strategies for biodiscovery and the virtually unexplored potential of lizard venoms in drug design and discovery.Bryan G. Fry, Kim Roelants, Kelly Winter, Wayne C. Hodgson, Laura Griesman, Hang Fai Kwok, Denis Scanlon, John Karas, Chris Shaw, Lily Wong and Janette A. Norma