Bacteria inhabiting spider webs enhance host silk extensibility.

Spider silk is a promising material with great potential in biomedical applications due to its incredible mechanical properties and resistance to degradation of commercially available bacterial strains. However, little is known about the bacterial communities that may inhabit spider webs and how these microorganisms interact with spider silk. In this study, we exposed two exopolysaccharide-secreting bacteria, isolated from webs of an orb spider, to major ampullate (MA) silk from host spiders. The naturally occurring lipid and glycoprotein surface layers of MA silk were experimentally removed to further probe the interaction between bacteria and silk. Extensibility of major ampullate silk produced by Triconephila clavata that was exposed to either Microbacterium sp. or Novosphigobium sp. was significantly higher than that of silk that was not exposed to bacteria (differed by 58.7%). This strain-enhancing effect was not observed when the lipid and glycoprotein surface layers of MA silks were removed. The presence of exopolysaccharides was detected through NMR from MA silks exposed to these two bacteria but not from those without exposure. Here we report for the first time that exopolysaccharide-secreting bacteria inhabiting spider webs can enhance extensibility of host MA silks and silk surface layers play a vital role in mediating such effects.

Buprofezin delayed the molting of Pardosa pseudoannulata, a predatory enemy for insect pests, by suppressing chitin synthase 1 expression.

Spiders, the major predatory enemies of insect pests in fields, are vulnerable to insecticides. In this study, we observed that the recommended dose of buprofezin delayed the molting of the pond wolf spider Pardosa pseudoannulata, although it had no lethal effect on the spiders. Since buprofezin is an insect chitin biosynthesis inhibitor, we identified two chitin synthase genes (PpCHS1 and PpCHS2) in P. pseudoannulata. Tissue-specific expression profiling showed that PpCHS1 was most highly expressed in cuticle. In contrast, PpCHS2 showed highest mRNA levels in the midgut and fat body. RNAi knockdown of PpCHS1 significantly delayed the molting of 12-days old spiderlings, whereas no significant effect on the molting was observed in the PpCHS2-silencing spiderlings. The expression of PpCHS1 was significantly suppressed in the spiderlings treated with buprofezin, but rescued by exogenous ecdysteroid ponasterone A (PA). Consistent with this result, the molting delay caused by buprofezin was also rescued by PA. The results revealed that buprofezin delayed the molting of spiders by suppressing PpCHS1 expression, which will benefit the protection of P. pseudoannulate and related spider species.

Identification of Peptides in Spider Venom Using Mass Spectrometry.

Spider venoms are composed of hundreds of proteins and peptides. Several of these venom toxins are cysteine-rich peptides in the mass range of 3-9 kDa. Small peptides (<3 kDa) can be fully characterized by mass spectrometry analysis, while proteins are generally identified by the bottom-up approach in which proteins are first digested with trypsin to generate shorter peptides for MS/MS characterization. In general, it is sufficient for protein identification to sequence two or more peptides, but for venom peptidomics it is desirable to completely elucidate peptide sequences and the number of disulfide bonds in the molecules. In this chapter, we describe a methodology to completely sequence and determine the number of disulfide bonds of spider venom peptides in the mass range of 3-9 kDa by multiple enzyme digestion, mass spectrometry of native and digested peptides, de novo analysis, and sequence overlap alignment.

Single-cell RNA sequencing of mid-to-late stage spider embryos: new insights into spider development.

BACKGROUND: The common house spider Parasteatoda tepidariorum represents an emerging new model organism of arthropod evolutionary and developmental (EvoDevo) studies. Recent technical advances have resulted in the first single-cell sequencing (SCS) data on this species allowing deeper insights to be gained into its early development, but mid-to-late stage embryos were not included in these pioneering studies. RESULTS: Therefore, we performed SCS on mid-to-late stage embryos of Parasteatoda and characterized resulting cell clusters by means of in-silico analysis (comparison of key markers of each cluster with previously published information on these genes). In-silico prediction of the nature of each cluster was then tested/verified by means of additional in-situ hybridization experiments with additional markers of each cluster. CONCLUSIONS: Our data show that SCS data reliably group cells with similar genetic fingerprints into more or less distinct clusters, and thus allows identification of developing cell types on a broader level, such as the distinction of ectodermal, mesodermal and endodermal cell lineages, as well as the identification of distinct developing tissues such as subtypes of nervous tissue cells, the developing heart, or the ventral sulcus (VS). In comparison with recent other SCS studies on the same species, our data represent later developmental stages, and thus provide insights into different stages of developing cell types and tissues such as differentiating neurons and the VS that are only present at these later stages.

High-strength and ultra-tough supramolecular polyamide spider silk fibers assembled via specific covalent and reversible hydrogen bonds.

Achieving ultra-high tensile strength and exceptional toughness is a longstanding goal for structural materials. However, previous attempts using covalent and non-covalent bonds have failed, leading to the belief that these two properties are mutually exclusive. Consequently, commercial fibers have been forced to compromise between tensile strength and toughness, as seen in the differences between nylon and Kevlar. To address this challenge, we drew inspiration from the disparate tensile strength and toughness of nylon and Kevlar, both of which are polyamide fibers, and developed an innovative approach that combines specific intermolecular disulfide bonds and reversible hydrogen bonds to create ultra-strong and ultra-tough polyamide spider silk fibers. Our resulting Supramolecular polyamide spider silk, which has a maximum molecular weight of 1084 kDa, exhibits high tensile strength (1180 MPa) and extraordinary toughness (433 MJ/m3), surpassing Kevlar's toughness 8-fold. This breakthrough presents a new opportunity for the sustainable development of spider silk as an environmentally friendly alternative to synthetic commercial fibers, as spider silk is composed of amino acids. Future research could explore the use of these techniques and fundamental knowledge to develop other super materials in various mechanical fields, with the potential to improve people's lives in many ways. STATEMENT OF SIGNIFICANCE: • By emulating synthetic commercial fibers such as nylon and polyethylene, we have successfully produced supramolecular-weight polyamide spider silk fibers with a molecular weight of 1084 kDa through a unique covalent bond-mediated linear polymerization reaction of spider silk protein molecules. This greatly surpasses the previous record of a maximum molecular weight of 556 kDa. • We obtained supramolecular polyamide spider silk fibers with both high-tensile strength and toughness. The stress at break is 1180 MPa, and the toughness is 8 times that of kevlar, reaching 433 MJ/m3. • Our results challenge the notion that it is impossible to manufacture fibers with both ultra-high tensile strength and ultra-toughness, and provide theoretical guidance for developing environmentally friendly and sustainable structural materials that meet industrial needs.

A Brief Overview of the Toxic Sphingomyelinase Ds of Brown Recluse Spider Venom and Other Organisms and Simple Methods To Detect Production of Its Signature Cyclic Ceramide Phosphate.

A special category of phospholipase D (PLD) in the venom of the brown recluse spider (Loxosceles reclusa) and several other sicariid spiders accounts for the dermonecrosis and many of the other clinical symptoms of envenomation. Related proteins are produced by other organisms, including fungi and bacteria. These PLDs are often referred to as sphingomyelinase Ds (SMase Ds) because they cleave sphingomyelin (SM) to choline and "ceramide phosphate." The lipid product has actually been found to be a novel sphingolipid: ceramide 1,3-cyclic phosphate (Cer1,3P). Since there are no effective treatments for the injury induced by the bites of these spiders, SMase D/PLDs are attractive targets for therapeutic intervention, and some of their features will be described in this minireview. In addition, two simple methods are described for detecting the characteristic SMase D activity using a fluorescent SM analog, (N-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-SM (C12-NBD-SM), that is cleaved to C12-NBD-Cer1,3P, which is easily separated from other potential metabolites by thin-layer chromatography and visualized under UV light. Besides confirming that C12-NBD-Cer1,3P is the only product detected upon incubation of C12-NBD-SM with brown recluse spider venom, the method was also able to detect for the first time very low levels of activity in venom from another spider, Kukulcania hibernalis The simplicity of the methods makes it relatively easy to determine this signature activity of SMase D/PLD. SIGNIFICANCE STATEMENT: The sphingomyelinase D/phospholipase D that are present in the venom of the brown recluse spider and other sources cause considerable human injury, but detection of the novel sphingolipid product, ceramide 1,3-cyclic phosphate, is not easy by previously published methods. This minireview describes simple methods for detection of this activity that will be useful for studies of its occurrence in spider venoms and other biological samples, perhaps including lesions from suspected spider bites and infections.

Regionalization of cell types in silk glands of Larinioides sclopetarius suggest that spider silk fibers are complex layered structures.

In order to produce artificial silk fibers with properties that match the native spider silk we likely need to closely mimic the spinning process as well as fiber architecture and composition. To increase our understanding of the structure and function of the different silk glands of the orb weaver Larinioides sclopetarius, we used resin sections for detailed morphology, paraffin embedded sections for a variety of different histological stainings, and a histochemical method for localization of carbonic anhydrase activity. Our results show that all silk glands, except the tubuliform glands, are composed of two or more columnar epithelial cell types, some of which have not been described previously. We observed distinct regionalization of the cell types indicating sequential addition of secretory products during silk formation. This means that the major ampullate, minor ampullate, aciniform type II, and piriform silk fibers most likely are layered and that each layer has a specific composition. Furthermore, a substance that stains positive for polysaccharides may be added to the silk in all glands except in the type I aciniform glands. Active carbonic anhydrase was found in all silk glands and/or ducts except in the type I aciniform and tubuliform glands, with the strongest staining in aggregate glands and their ductal nodules. Carbonic anhydrase plays an important role in the generation of a pH gradient in the major ampullate glands, and our results suggest that some other glands may also harbor pH gradients.

NMR assignment and dynamics of the dimeric form of soluble C-terminal domain major ampullate spidroin 2 from Latrodectus hesperus.

Spider dragline silk has attracted great interest due to its outstanding mechanical properties, which exceed those of man-made synthetic materials. Dragline silk, which is composed of at least major ampullate spider silk protein 1 and 2 (MaSp1 and MaSp2), contains a long repetitive domain flanked by N-terminal and C-terminal domains (NTD and CTD). Despite the small size of the CTD, this domain plays a crucial role as a molecular switch that regulates and directs spider silk self-assembly. In this study, we report the 1H, 13C, and 15N chemical shift assignments of the Latrodectus hesperus MaSp2 CTD in dimeric form at pH 7. Our solution NMR data demonstrated that this protein contains five helix regions connected by a flexible linker.

Domain swap facilitates structural transitions of spider silk protein C-terminal domains.

Domain swap is a mechanism of protein dimerization where the two interacting domains exchange parts of their structure. Web spiders make use of the process in the connection of C-terminal domains (CTDs) of spidroins, the soluble protein building blocks that form tough silk fibers. Besides providing connectivity and solubility, spidroin CTDs are responsible for inducing structural transitions during passage through an acidified assembly zone within spinning ducts. The underlying molecular mechanisms are elusive. Here, we studied the folding of five homologous spidroin CTDs from different spider species or glands. Four of these are domain-swapped dimers formed by five-helix bundles from spidroins of major and minor ampullate glands. The fifth is a dimer that lacks domain swap, formed by four-helix bundles from a spidroin of a flagelliform gland. Spidroins from this gland do not undergo structural transitions whereas the others do. We found a three-state mechanism of folding and dimerization that was conserved across homologues. In chemical denaturation experiments the native CTD dimer unfolded to a dimeric, partially structured intermediate, followed by full unfolding to denatured monomers. The energetics of the individual folding steps varied between homologues. Contrary to the common belief that domain swap stabilizes protein assemblies, the non-swapped homologue was most stable and folded four orders of magnitude faster than a swapped variant. Domain swap of spidroin CTDs induces an entropic penalty to the folding of peripheral helices, thus unfastening them for acid-induced unfolding within a spinning duct, which primes them for refolding into alternative structures during silk formation.

Role of ErbB and IL-1 signaling pathways in the dermonecrotic lesion induced by Loxosceles sphingomyelinases D.

Sphingomyelinase D (SMase D), the main toxic component of Loxosceles venom, has a well-documented role on dermonecrotic lesion triggered by envenomation with these species; however, the intracellular mechanisms involved in this event are still poorly known. Through differential transcriptomics of human keratinocytes treated with L. laeta or L. intermedia SMases D, we identified 323 DEGs, common to both treatments, as well as upregulation of molecules involved in the IL-1 and ErbB signaling. Since these pathways are related to inflammation and wound healing, respectively, we investigated the relative expression of some molecules related to these pathways by RT-qPCR and observed different expression profiles over time. Although, after 24 h of treatment, both SMases D induced similar modulation of these pathways in keratinocytes, L. intermedia SMase D induced earlier modulation compared to L. laeta SMase D treatment. Positive expression correlations of the molecules involved in the IL-1 signaling were also observed after SMases D treatment, confirming their inflammatory action. In addition, we detected higher relative expression of the inhibitor of the ErbB signaling pathway, ERRFI1, and positive correlations between this molecule and pro-inflammatory mediators after SMases D treatment. Thus, herein, we describe the cell pathways related to the exacerbation of inflammation and to the failure of the wound healing, highlighting the contribution of the IL-1 signaling pathway and the ERRFI1 for the development of cutaneous loxoscelism.

Natural spider silk nanofibrils produced by assembling molecules or disassembling fibers.

Spider silk is biocompatible, biodegradable, and rivals some of the best synthetic materials in terms of strength and toughness. Despite extensive research, comprehensive experimental evidence of the formation and morphology of its internal structure is still limited and controversially discussed. Here, we report the complete mechanical decomposition of natural silk fibers from the golden silk orb-weaver Trichonephila clavipes into ≈10 nm-diameter nanofibrils, the material's apparent fundamental building blocks. Furthermore, we produced nanofibrils of virtually identical morphology by triggering an intrinsic self-assembly mechanism of the silk proteins. Independent physico-chemical fibrillation triggers were revealed, enabling fiber assembly from stored precursors "at-will". This knowledge furthers the understanding of this exceptional material's fundamentals, and ultimately, leads toward the realization of silk-based high-performance materials. STATEMENT OF SIGNIFICANCE: Spider silk is one of the strongest and toughest biomaterials, rivaling the best man-made materials. The origins of these traits are still under debate but are mostly attributed to the material's intriguing hierarchical structure. Here we fully disassembled spider silk into 10 nm-diameter nanofibrils for the first time and showed that nanofibrils of the same appearance can be produced via molecular self-assembly of spider silk proteins under certain conditions. This shows that nanofibrils are the key structural elements in silk and leads toward the production of high-performance future materials inspired by spider silk.

Intersexual Differences in the Gene Expression of Phoneutria depilata (Araneae, Ctenidae) Toxins Revealed by Venom Gland Transcriptome Analyses.

The wandering spider, Phoneutria depilata, is one of Colombia's most active nocturnal arthropod predators of vertebrates and invertebrates. Its venom has been a relevant subject of study in the last two decades. However, the scarcity of transcriptomic data for the species limits our knowledge of the distinct components present in its venom for linking the mainly neurotoxic effects of the spider venom to a particular molecular target. The transcriptome of the P. depilata venom gland was analyzed to understand the effect of different diets or sex and the impact of these variables on the composition of the venom. We sequenced venom glands obtained from ten males and ten females from three diet treatments: (i) invertebrate: Tenebrio molitor, (ii) vertebrate: Hemidactylus frenatus, and (iii) mixed (T. molitor + H. frenatus). Of 17,354 assembled transcripts from all samples, 65 transcripts relating to venom production differed between males and females. Among them, 36 were classified as neurotoxins, 14 as serine endopeptidases, 11 as other proteins related to venom production, three as metalloprotease toxins, and one as a venom potentiator. There were no differences in transcripts across the analyzed diets, but when considering the effect of diets on differences between the sexes, 59 transcripts were differentially expressed. Our findings provide essential information on toxins differentially expressed that can be related to sex and the plasticity of the diet of P. depilata and thus can be used as a reference for venomics of other wandering spider species.

Global analysis of kinetics reveals the role of secondary nucleation in recombinant spider silk self-assembly.

Recombinant spider silk proteins can be prepared in scalable fermentation processes and have been proven as sources of biomaterials for biomedical and technical applications. Nanofibrils, formed through the self-assembly of these proteins, possess unique structural and mechanical properties, serving as fundamental building blocks for the fabrication of micro- and nanostructured scaffolds. Despite significant progress in utilizing nanofibrils-based morphologies of recombinant spider silk proteins, a comprehensive understanding of the molecular mechanisms of nanofibrils self-assembly remains a challenge. Here, a detailed kinetic study of nanofibril formation from a recombinant spider silk protein eADF4(C16) in dependence on the protein concentration, seeding, and temperature is provided. For the global fitting of kinetic data obtained during the fibril formation, we utilized the online platform AmyloFit. Evaluation of the data revealed that the self-assembly mechanism of recombinant spider silk is dominated by secondary nucleation. Thermodynamic analyses show that both primary and secondary nucleations, as well as the elongation step of the eADF4(C16), are endothermic processes.

Spider Silk Protein Forms Amyloid-Like Nanofibrils through a Non-Nucleation-Dependent Polymerization Mechanism.

Amyloid fibrils-nanoscale fibrillar aggregates with high levels of order-are pathogenic in some today incurable human diseases; however, there are also many physiologically functioning amyloids in nature. The process of amyloid formation is typically nucleation-elongation-dependent, as exemplified by the pathogenic amyloid-ß peptide (Aß) that is associated with Alzheimer's disease. Spider silk, one of the toughest biomaterials, shares characteristics with amyloid. In this study, it is shown that forming amyloid-like nanofibrils is an inherent property preserved by various spider silk proteins (spidroins). Both spidroins and Aß capped by spidroin N- and C-terminal domains, can assemble into macroscopic spider silk-like fibers that consist of straight nanofibrils parallel to the fiber axis as observed in native spider silk. While Aß forms amyloid nanofibrils through a nucleation-dependent pathway and exhibits strong cytotoxicity and seeding effects, spidroins spontaneously and rapidly form amyloid-like nanofibrils via a non-nucleation-dependent polymerization pathway that involves lateral packing of fibrils. Spidroin nanofibrils share amyloid-like properties but lack strong cytotoxicity and the ability to self-seed or cross-seed human amyloidogenic peptides. These results suggest that spidroins´ unique primary structures have evolved to allow functional properties of amyloid, and at the same time direct their fibrillization pathways to avoid formation of cytotoxic intermediates.

Peptides originally derived from Chilobrachys jingzhao tarantula venom possess beneficial effects on pancreatic beta cell health and function.

Clinical approval of the glucagon-like peptide-1 (GLP-1) mimetic exenatide for the treatment of type 2 diabetes highlights the therapeutic effectiveness of venom-derived peptides. In the present study, we examined and characterised the glucose-lowering potential of synthetic Jingzhaotoxin IX and Jingzhaotoxin XI peptides, which were originally isolated from the venom of the Chinese earth tarantula Chilobrachys jingzhao. Following confirmation of lack of beta-cell toxicity of synthetic peptides, assessment of enzymatic stability and effects on in vitro beta-cell function were studied, alongside putative mechanisms. Glucose homeostatic and appetite suppressive actions of Jingzhaotoxin IX and Jingzhaotoxin XI alone, or in combination with exenatide, were then assessed in normal overnight fasted C57BL/6 mice. Synthetic Jingzhaotoxin peptides were non-toxic and exhibited a decrease in mass of 6 Da in Krebs-Ringer bicarbonate buffer suggesting inhibitor cysteine knot (ICK)-like formation, but interestingly were liable to plasma enzyme degradation. The Jingzhaotoxin peptides evoked prominent insulin secretion from BRIN BD11 beta-cells, with activity somewhat characteristic of Kv2.1 channel binding. In addition, Jingzhaotoxin peptides enhanced beta-cell proliferation and provided significant protection against cytokine-induced apoptosis. When injected co-jointly with glucose, the Jingzhaotoxin peptides slightly decreased blood-glucose levels but had no effect on appetite in overnight fasted mice. Whilst the Jingzhaotoxin peptides did not enhance exenatide-induced benefits on glucose homeostasis, they augmented exenatide-mediated suppression of appetite. Taken together, these data highlight the therapeutic potential of tarantula venom-derived peptides, such as Jingzhaotoxin IX and Jingzhaotoxin XI either alone or in combination with exenatide, for diabetes and related obesity.

pH-dependent self-assembly mechanism of a single repetitive domain from a spider silk protein.

Spider silk is self-assembled from full-length silk proteins, and some silk protein fragments can also form silk-like fibers in vitro. However, the mechanism underlying the silk fiber formation is not understood well. In this study, we investigated the fiber formation of a single repetitive domain (RP) from a minor ampullate silk protein (MiSp). Our findings revealed that pH and salt concentration affect not only the stability of MiSp-RP but also its self-assembly into fibers and aggregates. Using nuclear magnetic resonance (NMR) spectroscopy, we solved the three-dimensional (3D) structure of MiSp RP in aqueous solution. On the basis of the structure and mutagenesis, we revealed that charge-dipole interactions are responsible for the pH- and salt-dependent properties of MiSp-RP. Our results indicate that fiber formation is regulated by a delicate balance between intermolecular and intramolecular interactions, rather than by the protein stability alone. These findings have implications for the design of silk proteins for mass production of spider silk.

Biochemical mechanism involved in the enhancement of the Young's modulus of silk by the SpiCE protein.

Silk fibers are known for their superior mechanical properties, with the strongest possessing over seven times the toughness of kevlar. Recently, low molecular weight non-spidroin protein, spider-silk constituting element (SpiCE), has been reported to enhance the mechanical properties of silk; however, its specific action mechanism has not yet been elucidated. Here, we explored the mechanism by which SpiCE strengthened the mechanical properties of major ampullate spidroin 2 (MaSp2) silk through hydrogen bonds and salt bridges of the silk structure via all-atom molecular dynamics simulations. Tensile pulling simulation on silk fiber with SpiCE protein revealed that the SpiCE protein enhanced the Young's modulus by up to 40% more than that of the wild type. Bond characteristic analysis revealed that SpiCE and MaSp2 formed more hydrogen bonds and salt bridges than the MaSp2 wild-type model. Sequence analysis of MaSp2 silk fiber and SpiCE protein revealed that SpiCE protein contained more amino acids that could act as hydrogen bond acceptors/donors and salt bridge partners. Our results provide insights into the mechanism by which non-spidroin proteins strengthen the properties of silk fibers and lay the groundwork for the development of material selection criteria for the design of de novo artificial silk fibers.

Response of the Pardosa astrigera bacterial community to Cry1B protein.

While genetically modified (GM) crops bring economic benefits to human beings, their impact on non-target organisms has become an important part of environmental safety assessments. Symbiotic bacteria play an important role in eukaryotic biological functions and can adjust host communities to adapt to new environments. Therefore, this study examined the effects of Cry1B protein on the growth and development of non-target natural enemies of Pardosa astrigera (L. Koch) from the perspective of symbiotic bacteria. Cry1B protein had no significant effect on the health indicators of P. astrigera (adults and 2nd instar spiderlings). 16S rRNA sequencing results revealed that Cry1B protein did not change the symbiotic bacteria species composition of P. astrigera, but did reduce the number of OTU and species diversity. In 2nd instar spiderlings, neither the dominant phylum (Proteobacteria) nor the dominant genus (Acinetobacter) changed, but the relative abundance of Corynebacterium-1 decreased significantly; in adult spiders, the dominant bacteria genera of females and males were different. The dominant bacterial genera were Brevibacterium in females and Corynebacterium-1 in males, but Corynebacterium-1 was the dominant bacteria in both females and males feeding on Cry1B. The relative abundance of Wolbachia also increased significantly. In addition, bacteria in other genera varied significantly by sex. KEGG results showed that Cry1B protein only altered the significant enrichment of metabolic pathways in female spiders. In conclusion, the effects of Cry1B protein on symbiotic bacteria vary by growth and development stage and sex.

Predicting mechanical properties of silk from its amino acid sequences via machine learning.

The silk fiber is increasingly being sought for its superior mechanical properties, biocompatibility, and eco-friendliness, making it promising as a base material for various applications. One of the characteristics of protein fibers, such as silk, is that their mechanical properties are significantly dependent on the amino acid sequence. Numerous studies have been conducted to determine the specific relationship between the amino acid sequence of silk and its mechanical properties. Still, the relationship between the amino acid sequence of silk and its mechanical properties is yet to be clarified. Other fields have adopted machine learning (ML) to establish a relationship between the inputs, such as the ratio of different input material compositions and the resulting mechanical properties. We have proposed a method to convert the amino acid sequence into numerical values for input and succeeded in predicting the mechanical properties of silk from its amino acid sequences. Our study sheds light on predicting mechanical properties of silk fiber from respective amino acid sequences.

Unusual pKa Values Mediate the Self-Assembly of Spider Dragline Silk Proteins.

Spider dragline silk is a remarkably tough biomaterial and composed primarily of spidroins MaSp1 and MaSp2. During fiber self-assembly, the spidroin N-terminal domains (NTDs) undergo rapid dimerization in response to a pH gradient. However, obtaining a detailed understanding of this mechanism has been hampered by a lack of direct evidence regarding the protonation states of key ionic residues. Here, we elucidated the solution structures of MaSp1 and MaSp2 NTDs from Trichonephila clavipes and determined the experimental pKa values of conserved residues involved in dimerization using NMR. Surprisingly, we found that the Asp40 located on an acidic cluster protonates at an unusually high pH (∼6.5-7.1), suggesting the first step in the pH response. Then, protonation of Glu119 and Glu79 follows, with pKas above their intrinsic values, contributing toward stable dimer formation. We propose that exploiting the atypical pKa values is a strategy to achieve tight spatiotemporal control of spider silk self-assembly.