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Spider silk's dual identity Many proteins form fibrillar structures at high concentrations, but spider silk proteins, with highly repetitive segments flanked by non-repetitive (NR) terminal domains, behave differently. They are remarkably soluble when stored at high concentration yet can convert to extremely sturdy fibres on demand. The molecular mechanism that makes this possible is not yet clear, but two structural studies in this issue provide new clues. Askarieh et al . present the 1.7 Å X-ray crystal structure of the N-terminal domain of a dragline spidroin from the nursery web spider Euprosthenops australis . The structure shows how this highly conserved domain can regulate silk assembly by preventing premature aggregation of spidroins and triggering polymerization as the pH falls along the silk extrusion duct. Hagn et al . determined the solution structure of the C-terminal NR domain of the dragline silk protein fibroin 3 from the common orb-weaver Araneus diadematus . They observe a conformational switch, activated by chemical or mechanical stimuli, between storage and assembly forms of the protein. Spider silk proteins are remarkably soluble when stored at high concentration and yet can be converted to extremely sturdy fibres, through unknown molecular mechanisms. Here, the X-ray structure of the amino-terminal domain of a silk protein is presented, revealing how evolutionarily conserved polar surfaces might control self-assembly as the pH is lowered along the spider's silk extrusion duct. Such a mechanism might be applicable to the design of versatile fibrous materials. Nature’s high-performance polymer, spider silk, consists of specific proteins, spidroins, with repetitive segments flanked by conserved non-repetitive domains 1 , 2 . Spidroins are stored as a highly concentrated fluid dope. On silk formation, intermolecular interactions between repeat regions are established that provide strength and elasticity 3 , 4 . How spiders manage to avoid premature spidroin aggregation before self-assembly is not yet established. A pH drop to 6.3 along the spider’s spinning apparatus, altered salt composition and shear forces are believed to trigger the conversion to solid silk, but no molecular details are known. Miniature spidroins consisting of a few repetitive spidroin segments capped by the carboxy-terminal domain form metre-long silk-like fibres irrespective of pH 5 . We discovered that incorporation of the amino-terminal domain of major ampullate spidroin 1 from the dragline of the nursery web spider Euprosthenops australis (NT) into mini-spidroins enables immediate, charge-dependent self-assembly at pH values around 6.3, but delays aggregation above pH 7. The X-ray structure of NT, determined to 1.7 Å resolution, shows a homodimer of dipolar, antiparallel five-helix bundle subunits that lack homologues. The overall dimeric structure and observed charge distribution of NT is expected to be conserved through spider evolution and in all types of spidroins. Our results indicate a relay-like mechanism through which the N-terminal domain regulates spidroin assembly by inhibiting precocious aggregation during storage, and accelerating and directing self-assembly as the pH is lowered along the spider’s silk extrusion duct.

Acid-sensing ion channels (ASICs) are voltage-independent, amiloride-sensitive channels involved in diverse physiological processes ranging from nociception to taste. Despite the importance of ASICs in physiology, we know little about the mechanism of channel activation. Here we show that psalmotoxin activates non-selective and Na + -selective currents in chicken ASIC1a at pH 7.25 and 5.5, respectively. Crystal structures of ASIC1a–psalmotoxin complexes map the toxin binding site to the extracellular domain and show how toxin binding triggers an expansion of the extracellular vestibule and stabilization of the open channel pore. At pH 7.25 the pore is approximately 10 Å in diameter, whereas at pH 5.5 the pore is largely hydrophobic and elliptical in cross-section with dimensions of approximately 5 by 7 Å, consistent with a barrier mechanism for ion selectivity. These studies define mechanisms for activation of ASICs, illuminate the basis for dynamic ion selectivity and provide the blueprints for new therapeutic agents. Acid-sensing ion channels (ASICs) are voltage-independent ion channels that participate in a broad range of biological processes, including nociception and mechanosensation; here X-ray crystal structures of the complexes of chicken ASIC1a with psalmotoxin, a peptide toxin from tarantula, indicate that toxin binding triggers an expansion of the extracellular vestibule and stabilization of the open channel pore. Open ASIC1a channel structure Acid-sensing ion channels (ASICs) are members of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily of voltage-independent ion channels. ENaCs, including ASICs, participate in a broad range of biological processes, such as nociception, mechanosensation and regulation of sodium-ion homeostasis. Here, Isabelle Baconguis and Eric Gouaux show that psalmotoxin, a peptide toxin from the tarantula, activates nonselective and sodium-selective currents in chicken ASIC1a. X-ray crystal structures of the chicken ASIC1a–psalmotoxin complexes indicate that toxin binding triggers an expansion of the extracellular vestibule and stabilization of the open channel pore. This view of an important type of ion channel in an open conformation is of relevance to the design of open-channel blockers that might have therapeutic promise for the treatment of pain.

As major components of spider venoms, neurotoxic peptides exhibit structural diversity, target specificity, and have great pharmaceutical potential. Deep learning may be an alternative to the laborious and time-consuming methods for identifying these peptides. However, the major hurdle in developing a deep learning model is the limited data on neurotoxic peptides. Here, we present a peptide data augmentation method that improves the recognition of neurotoxic peptides via a convolutional neural network model. The neurotoxic peptides were augmented with the known neurotoxic peptides from UniProt database, and the models were trained using a training set with or without the generated sequences to verify the augmented data. The model trained with the augmented dataset outperformed the one with the unaugmented dataset, achieving accuracy of 0.9953, precision of 0.9922, recall of 0.9984, and F1 score of 0.9953 in simulation dataset. From the set of all RNA transcripts of Callobius koreanus spider, we discovered neurotoxic peptides via the model, resulting in 275 putative peptides of which 252 novel sequences and only 23 sequences showing homology with the known peptides by Basic Local Alignment Search Tool. Among these 275 peptides, four were selected and shown to have neuromodulatory effects on the human neuroblastoma cell line SH-SY5Y. The augmentation method presented here may be applied to the identification of other functional peptides from biological resources with insufficient data.

The venoms of spiders from the RTA (retro-lateral tibia apophysis) clade contain diverse short linear peptides (SLPs) that offer a rich source of therapeutic candidates. Many of these peptides have insecticidal, antimicrobial and/or cytolytic activities, but their biological functions are unclear. Here, we explore the bioactivity of all known members of the A-family of SLPs previously identified in the venom of the Chinese wolf spider (Lycosa shansia). Our broad approach included an in silico analysis of physicochemical properties and bioactivity profiling for cytotoxic, antiviral, insecticidal and antibacterial activities. We found that most members of the A-family can form α-helices and resemble the antibacterial peptides found in frog poison. The peptides we tested showed no cytotoxic, antiviral or insecticidal activities but were able to reduce the growth of bacteria, including clinically relevant strains of Staphylococcus epidermidis and Listeria monocytogenes. The absence of insecticidal activity may suggest that these peptides have no role in prey capture, but their antibacterial activity may help to defend the venom gland against infection.

The potent neurotoxic venom of the black widow spider contains a cocktail of seven phylum-specific latrotoxins (LTXs), but only one, α-LTX, targets vertebrates. This 130 kDa toxin binds to receptors at presynaptic nerve terminals and triggers a massive release of neurotransmitters. It is widely accepted that LTXs tetramerize and insert into the presynaptic membrane, thereby forming Ca 2+ -conductive pores, but the underlying mechanism remains poorly understood. LTXs are homologous and consist of an N-terminal region with three distinct domains, along with a C-terminal domain containing up to 22 consecutive ankyrin repeats. Here we report cryoEM structures of the vertebrate-specific α-LTX tetramer in its prepore and pore state. Our structures, in combination with AlphaFold2-based structural modeling and molecular dynamics simulations, reveal dramatic conformational changes in the N-terminal region of the complex. Four distinct helical bundles rearrange and together form a highly stable, 15 nm long, cation-impermeable coiled-coil stalk. This stalk, in turn, positions an N-terminal pair of helices within the membrane, thereby enabling the assembly of a cation-permeable channel. Taken together, these data give insight into a unique mechanism for membrane insertion and channel formation, characteristic of the LTX family, and provide the necessary framework for advancing novel therapeutics and biotechnological applications. Black widow spider venom contains seven structurally related toxins acting at presynaptic nerve terminals. Here, the authors provide cryo-EM structures of vertebrate-specific αLTX in two states and decipher how the toxin forms cation-selective pores.

Latrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (α-latrotoxin (α-LTX)), five specialized on insects (α, β, γ, δ, ε- latroinsectotoxins (LITs), and one on crustaceans (α-latrocrustatoxin (α-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca 2+ -conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear. Here, we report cryoEM structures of the α-LCT monomer and the δ-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel α-helices. Both domains are flexibly linked via an N-terminal α-helical domain and a small β-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant α-LCT and δ-LIT form channels in artificial membrane bilayers, that are stabilized by Ca 2+ ions and allow calcium flux at negative membrane potentials. Our comparative analysis between α-LCT and δ-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family. The venom of Latrodectus spiders contains seven Latrotoxins (LaTXs), among them α-latrocrustatoxin (LCT) and δ- latroinsectotoxins δ-LIT. LaTXs bind to specific receptors on the surface of neuronal cells and target the molecular exocytosis machinery. Here, the authors present the cryo-EM structure of the α-LCT monomer and the δ-LIT dimer, which reveal that LaTXs are organized in four domains and they discuss the potential oligomerisation mechanism that takes place before LaTXs membrane insertion. Both recombinant α-LCT and δ-LIT form channels in artificial membrane bilayers, that are stabilized by Ca 2+ ions.

Bites of Loxosceles spiders can lead to a set of clinical manifestations called loxoscelism, and are considered a public health problem in many regions. The signs and symptoms of loxoscelism are divided into cutaneous and systemic forms. The former is more frequent and includes signs of envenoming at the bite site or neighboring regions. Systemic loxoscelism, although much less frequent, is associated with complications, and can even lead to death. It may include intravascular hemolysis, acute renal failure, and thrombocytopenia. Loxosceles venoms are enriched with phospholipases D (PLDs), which are a family of isoforms found at intra-species and inter-species levels. Under experimental conditions, these enzymes reproduce the main clinical signs of loxoscelism, including an exacerbated inflammatory response at the bite site and dermonecrosis, as well as thrombocytopenia, intravascular hemolysis, and acute renal failure. The role of PLDs in cutaneous loxoscelism was described over forty years ago, when studies identified and purified toxins featured as sphingomyelinase D. More recently, the production of recombinant PLDs and discoveries about their structure and mechanism has enabled a deeper characterization of these enzymes. In this review, we describe these biochemical and functional features of Loxosceles PLDs that determine their involvement in systemic loxoscelism.

Prey-specialised spiders are adapted to capture specific prey items, including dangerous prey. The venoms of specialists are often prey-specific and less complex than those of generalists, but their venom composition has not been studied in detail. Here, we investigated the venom of the prey-specialised white-tailed spiders (Lamponidae: Lampona ), which utilise specialised morphological and behavioural adaptations to capture spider prey. We analysed the venom composition using proteo-transcriptomics and taxon-specific toxicity using venom bioassays. Our analysis identified 208 putative toxin sequences, comprising 103 peptides < 10 kDa and 105 proteins > 10 kDa. Most peptides belonged to one of two families characterised by scaffolds containing eight or ten cysteine residues. Toxin-like proteins showed similarity to galectins, leucine-rich repeat proteins, trypsins and neprilysins. The venom of Lampona was shown to be more potent against the preferred spider prey than against alternative cricket prey. In contrast, the venom of a related generalist was similarly potent against both prey types. These data provide insights into the molecular adaptations of venoms produced by prey-specialised spiders.

A taste for pain Three peptides isolated from the venom of the West Indian tarantula Psalmopoeus cambridgei have been found to promote pain and inflammation by activating the same neuronal receptor as capsaicin, the hot component of chilli peppers. This suggests that tarantulas and chillis use similar tactics to deter predators. The newly discovered peptides are also unusual because they trigger an excitatory response. Peptides with similar structures that bind to other ion channels are already known, but are inhibitory. Bites and stings from venomous creatures can produce pain and inflammation as part of their defensive strategy to ward off predators or competitors 1 , 2 . Molecules accounting for lethal effects of venoms have been extensively characterized, but less is known about the mechanisms by which they produce pain. Venoms from spiders, snakes, cone snails or scorpions contain a pharmacopoeia of peptide toxins that block receptor or channel activation as a means of producing shock, paralysis or death 3 , 4 , 5 . We examined whether these venoms also contain toxins that activate (rather than inhibit) excitatory channels on somatosensory neurons to produce a noxious sensation in mammals. Here we show that venom from a tarantula that is native to the West Indies contains three inhibitor cysteine knot (ICK) peptides that target the capsaicin receptor (TRPV1), an excitatory channel expressed by sensory neurons of the pain pathway 6 . In contrast with the predominant role of ICK toxins as channel inhibitors 5 , 7 , these previously unknown ‘vanillotoxins’ function as TRPV1 agonists, providing new tools for understanding mechanisms of TRP channel gating. Some vanillotoxins also inhibit voltage-gated potassium channels, supporting potential similarities between TRP and voltage-gated channel structures. TRP channels can now be included among the targets of peptide toxins, showing that animals, like plants (for example, chilli peppers), avert predators by activating TRP channels on sensory nerve fibres to elicit pain and inflammation.

The inhibitor cystine knot (ICK) is an unusual three-disulfide architecture in which one of the disulfide bonds bisects a loop formed by the two other disulfide bridges and the intervening sections of the protein backbone. Peptides containing an ICK motif are frequently considered to have high levels of thermal, chemical and enzymatic stability due to cross-bracing provided by the disulfide bonds. Experimental studies supporting this contention are rare, in particular for spider-venom toxins, which represent the largest diversity of ICK peptides. We used ω-hexatoxin-Hv1a (Hv1a), an insecticidal toxin from the deadly Australian funnel-web spider, as a model system to examine the contribution of the cystine knot to the stability of ICK peptides. We show that Hv1a is highly stable when subjected to temperatures up to 75 °C, pH values as low as 1, and various organic solvents. Moreover, Hv1a was highly resistant to digestion by proteinase K and when incubated in insect hemolymph and human plasma. We demonstrate that the ICK motif is essential for the remarkable stability of Hv1a, with the peptide’s stability being dramatically reduced when the disulfide bonds are eliminated. Thus, this study demonstrates that the ICK motif significantly enhances the chemical and thermal stability of spider-venom peptides and provides them with a high level of protease resistance. This study also provides guidance to the conditions under which Hv1a could be stored and deployed as a bioinsecticide.