Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface.

Spider dragline silk is a natural polymer harboring unique physical and biochemical properties that make it an ideal biomaterial. Artificial silk production requires an understanding of the in vivo mechanisms spiders use to convert soluble proteins, called spidroins, into insoluble fibers. Controlled dimerization of the spidroin N-terminal domain (NTD) is crucial to this process. Here, we report the crystal structure of the Nephila clavipes major ampullate spidroin NTD dimer. Comparison of our N. clavipes NTD structure with previously determined Euprosthenops australis NTD structures reveals subtle conformational alterations that lead to differences in how the subunits are arranged at the dimer interface. We observe a subset of contacts that are specific to each ortholog, as well as a substantial increase in asymmetry in the interactions observed at the N. clavipes NTD dimer interface. These asymmetric interactions include novel intermolecular salt bridges that provide new insights into the mechanism of NTD dimerization. We also observe a unique intramolecular "handshake" interaction between two conserved acidic residues that our data suggest adds an additional layer of complexity to the pH-sensitive relay mechanism for NTD dimerization. The results of a panel of tryptophan fluorescence dimerization assays probing the importance of these interactions support our structural observations. Based on our findings, we propose that conformational selectivity and plasticity at the NTD dimer interface play a role in the pH-dependent transition of the NTD from monomer to stably associated dimer as the spidroin progresses through the silk extrusion duct.

Polyelectrolyte Fiber Assembly of Plant-Derived Spider Silk-like Proteins.

Spider dragline silk is a proteinaceous material that combines superior toughness and biocompatibility, which makes it a promising biomaterial. The distinct protein structure and the fiber formation process contribute to the superior toughness of dragline silk. Previously, we have produced recombinant spider silk-like proteins in transgenic tobacco that are readily purified from plant extracts. The plant-derived spidroin-like proteins consisted of native major ampullate spidroin 1 or spidroin 2 N- and C-termini flanking 8, 16, or 32 copies of their respective consensus block repeats (mini-spidroins). Here, we present the generation of fibers from mini-spidroins (rMaSp1R8 and rMaSp2R8) by polyelectrolyte complex formation using an anionic polyelectrolyte, gellan gum. Mini-spidroins, when treated with acetic acid and cross-linked by glutaraldehyde, formed a thin film at the interface when overlaid with a gellan gum solution. Immediate pulling of the film resulted in autofluorescent fibrous materials from either mini-spidroin alone or a combination of rMaSp1R8 and rMaSp2R8 (70:30). Addition of chitosan to the mini-spidroin solutions permitted continuous fiber production until the spinning dope supply was exhausted. When air-dried as-spun fibers were rehydrated and stretched in water, the fiber diameter decreased and the overall toughness improved. This study showed that spider silk-like fibers can be produced in large quantities through charge attraction that assembles chitosan, mini-spidroins, and gellan gum into fibrous complexes. We speculate that the spider silk self-assembly process in the duct may involve attraction of variously charged chitinous polymers, spidroins, and glycoproteins.

Spider silk-like proteins derived from transgenic Nicotiana tabacum.

The high tensile strength and biocompatibility of spider dragline silk makes it a desirable material in many engineering and tissue regeneration applications. Here, we present the feasibility to produce recombinant proteins in transgenic tobacco Nicotiana tabacum with sequences representing spider silk protein building blocks . Recombinant mini-spidroins contain native N- and C-terminal domains of major ampullate spidroin 1 (rMaSp1) or rMaSp2 flanking an abbreviated number (8, 16 or 32) of consensus repeat domains. Two different expression plasmid vectors were tested and a downstream chitin binding domain and self-cleavable intein were included to facilitate protein purification. We confirmed gene insertion and RNA transcription by PCR and reverse-transcriptase PCR, respectively. Mini-spidroin production was detected by N-terminus specific antibodies. Purification of mini-spidroins was performed through chitin affinity chromatography and subsequent intein activation with reducing reagent. Mini-spidroins, when dialyzed and freeze-dried, formed viscous gelatin-like fluids.

Spidroin N-terminal domain promotes a pH-dependent association of silk proteins during self-assembly.

Spider silks are spun from concentrated solutions of spidroin proteins. The appropriate timing of spidroin assembly into organized fibers must be highly regulated to avoid premature fiber formation. Chemical and physical signals presented to the silk proteins as they pass from the ampulle and through the tapered duct include changes in ionic environment and pH as well as the introduction of shear forces. Here, we show that the N-terminal domain of spidroins from the major ampullate gland (MaSp-NTDs) for both Nephila and Latrodectus spiders associate noncovalently as homodimers. The MaSp-NTDs are highly pH-responsive and undergo a structural transition in the physiological pH range of the spider duct. Tryptophan fluorescence of the MaSp-NTDs reveals a change in conformation when pH is decreased, and the pH at which the transition occurs is determined by the amount and type of salt present. Size exclusion chromatography and pulldown assays both indicate that the lower pH conformation is associated with a significantly increased MaSp-NTD homodimer stability. By transducing the duct pH signal into specific protein-protein interactions, this conserved spidroin domain likely contributes significantly to the silk-spinning process. Based on these results, we propose a model of spider silk assembly dynamics as mediated through the MaSp-NTD.

Corrigendum: Advances in Plant-Derived Scaffold Proteins.

[This corrects the article DOI: 10.3389/fpls.2020.00122.].

Advances in Plant-Derived Scaffold Proteins.

Scaffold proteins form critical biomatrices that support cell adhesion and proliferation for regenerative medicine and drug screening. The increasing demand for such applications urges solutions for cost effective and sustainable supplies of hypoallergenic and biocompatible scaffold proteins. Here, we summarize recent efforts in obtaining plant-derived biosynthetic spider silk analogue and the extracellular matrix protein, collagen. Both proteins are composed of a large number of tandem block repeats, which makes production in bacterial hosts challenging. Furthermore, post-translational modification of collagen is essential for its function which requires co-transformation of multiple copies of human prolyl 4-hydroxylase. We discuss our perspectives on how the GAANTRY system could potentially assist the production of native-sized spider dragline silk proteins and prolyl hydroxylated collagen. The potential of recombinant scaffold proteins in drug delivery and drug discovery is also addressed.

Seed dehydration and the establishment of desiccation tolerance during seed maturation is altered in the Arabidopsis thaliana mutant atem6-1.

The end of orthodox seed development is typified by a developmentally regulated period of dehydration leading to the loss of bulk water from the entire structure. When dehydration occurs, the cytoplasm condenses and intracellular components become more crowded, providing an environment amenable to numerous undesirable interactions that can lead to protein aggregation, denaturation and organelle-cell membrane fusion. Acquisition of desiccation tolerance, or the ability to withstand these very low water potentials and consequent molecular crowding, has been correlated with the accumulation of various protective compounds including proteins and sugars. Among these are the late embryogenesis abundant (LEA) proteins, a diverse class of highly abundant, heat-stable proteins that accumulate late in embryo maturation coincident with the acquisition of desiccation tolerance. Previous work led us to hypothesize that the protein ATEM6, one of the two Arabidopsis thaliana group 1 LEA proteins, is involved in regulating the rate at which water is lost from the maturing embryo; homozygous atem6-1 mutants display premature dehydration of seeds at the distal end of the silique. Here we demonstrate that rehydrated, mature seeds from atem6-1 mutant plants lose more water during subsequent air drying than wild-type seeds, consistent with a role for ATEM6 protein in water binding/loss during embryo maturation. In addition, and possibly as a result of premature dehydration, mutant seeds along the entire length of the silique acquire desiccation tolerance earlier than their wild-type counterparts. We further demonstrate precocious, and perhaps elevated, expression of the other A. thaliana group 1 LEA protein, ATEM1, that may compensate for loss or ATEM6 expression. However, this observation could also be consistent with acceleration of the entire normal maturation program in atem6-1 mutant embryos. Interestingly, ATEM6 protein does not appear to be required in mature seeds for viability or efficient germination.

A predicted N-terminal helical domain of a Group 1 LEA protein is required for protection of enzyme activity from drying.

Late embryogenesis abundant (LEA) proteins have been repeatedly implicated in the acquisition of desiccation tolerance in angiosperm seed embryos. However, the mechanism(s) by which protection occurs is not well understood. While the Group 1 LEA proteins are predicted to be largely unordered in solution, there is strong evidence that upon drying these proteins undergo a structural transition that leads to an increase in alpha-helical content. Several studies also suggest there is a direct interaction between Group 1 LEA proteins and other molecules in the cytoplasm that may be critical for the establishment of desiccation tolerance during embryo maturation. We have produced a recombinant Group 1 LEA protein and show that it is capable of protecting the enzyme lactate dehydrogenase from the deleterious effects of drying. We have also evaluated the ability of various altered recombinant Group 1 LEA proteins to protect in the same assay. Our results suggest that the highly conserved 20 amino acid Group 1 LEA signature motif is not required for protection in our in vitro assay. However, introduction of two juxtaposed proline residues into an N-terminal helical domain predicted to exist in the hydrated structure significantly compromises the ability of the recombinant protein to provide protection from drying. These results suggest that the N-terminal domain of Group 1 LEA proteins may be important for proper folding during dehydration.

Evaluating adhesion and alignment of dental pulp stem cells to a spider silk substrate for tissue engineering applications.

A proposed source of stem cells for nerve regeneration are dental pulp stem cells (DPSCs), based on their close embryonic origin to neurons and the ease with which DPSCs can be obtained from a donor. This study evaluated the response of human DPSCs to spider dragline silk fibers, a potential substrate material for tissue regeneration. The DPSCs' morphology and spread pattern were characterized after these cells were plated onto Nephila clavipes dragline fibers in media. In addition, the responses of two other well established cell lines, osteoblasts (7F2s), and fibroblasts (3T3s), were also studied under identical conditions. The inclusion of 3T3s and 7F2s in this study allowed for both direct comparisons to prior published work and a qualitative comparison to the morphology of the DPSCs. After twelve days, the DPSCs exhibited greater relative alignment and adherence to the spider dragline fibers than the 3T3s and 7F2s. The impact of a common sterilization method (ultraviolet light) on the spider dragline fiber surface and subsequent cell response to this modified surface was also characterized. Exposure of the silk to ultraviolet light did not have a measureable effect on cell alignment, but it did eliminate bacterial growth and changed fiber surface roughness. Spiders' exposure to stressful environments did not have an effect on silk to impair cell alignment or adhesion. Synthetic recombinant protein silk did not act as a substrate for cell adhesion or alignment but hydrogels with similar composition supported cell attachment, growth and proliferation. In all cases, natural drawn spider silk acted as an effective substrate for cellular adhesion and alignment of DPSCs and could be used in neural differentiation applications.

A Leishmania secretion system for the expression of major ampullate spidroin mimics.

Spider major ampullate silk fibers have been shown to display a unique combination of relatively high fracture strength and toughness compared to other fibers and show potential for tissue engineering scaffolds. While it is not possible to mass produce native spider silks, the potential ability to produce fibers from recombinant spider silk fibers could allow for an increased innovation rate within tissue engineering and regenerative medicine. In this pilot study, we improved upon a prior fabrication route by both changing the expression host and additives to the fiber pulling precursor solution to improve the performance of fibers. The new expression host for producing spidroin protein mimics, protozoan parasite Leishmania tarentolae, has numerous advantages including a relatively low cost of culture, rapid growth rate and a tractable secretion pathway. Tensile testing of hand pulled fibers produced from these spidroin-like proteins demonstrated that additives could significantly modify the fiber's mechanical and/or antimicrobial properties. Cross-linking the proteins with glutaraldehyde before fiber pulling resulted in a relative increase in tensile strength and decrease in ductility. The addition of ampicillin into the spinning solution resulted in the fibers being able to inhibit bacterial growth.

Recombinant Dragline Silk-Like Proteins-Expression and Purification.

Spider dragline silk is a proteinaceous fiber with impressive physical characteristics making it attractive for use in advanced materials. The fiber is composed of two proteins (spidroins MaSp1 and MaSp2), each of which contains a large central repeat array flanked by non-repetitive N- and C-terminal domains. The repeat arrays appear to be largely responsible for the tensile properties of the fiber, suggesting that the N- and C-terminal domains may be involved in self-assembly. We recently isolated the MaSp1 and MaSp2 N-terminal domains from Nephila clavipes and have incorporated these into mini-silk genes for expression in transgenic systems. Current efforts involve the development of expression vectors that will allow purification using a removable affinity tag for scalable protein purification.

NMR assignments of the N-terminal domain of Nephila clavipes spidroin 1.

The building blocks of spider dragline silk are two fibrous proteins secreted from the major ampullate gland named spidroins 1 and 2 (MaSp1, MaSp2). These proteins consist of a large central domain composed of approximately 100 tandem copies of a 35-40 amino acid repeat sequence. Non-repetitive N and C-terminal domains, of which the C-terminal domain has been implicated to transition from soluble and insoluble states during spinning, flank the repetitive core. The N-terminal domain until recently has been largely unknown due to difficulties in cloning and expression. Here, we report nearly complete assignment for all (1)H, (13)C, and (15)N resonances in the 14 kDa N-terminal domain of major ampullate spidroin 1 (MaSp1-N) of the golden orb-web spider Nephila clavipes.

The Arabidopsis group 1 LATE EMBRYOGENESIS ABUNDANT protein ATEM6 is required for normal seed development.

As part of the embryo maturation process, orthodox seeds undergo a developmentally regulated dehydration period. The LATE EMBRYOGENESIS ABUNDANT (LEA) genes encode a large and diverse family of proteins expressed during this time. Many hypothesize that LEA proteins act by mitigating water loss and maintaining cellular stability within the desiccated seed, although the mechanisms of their actions remain largely unknown. The model plant Arabidopsis (Arabidopsis thaliana) contains two genes belonging to the group 1 LEA family, ATEM1 and ATEM6, and knockout mutations in these genes are being sought as a means to better understand group 1 LEA protein function during embryo maturation. We have identified a T-DNA insertion allele of the ATEM6 gene in which the T-DNA is present just downstream of the protein coding region. While this gene is transcriptionally active and encodes a wild-type protein, there is no detectable ATEM6 protein in mature seeds. Mutant seeds display premature seed dehydration and maturation at the distal end of siliques, demonstrating that this protein is required for normal seed development. We propose that one function for group 1 LEA proteins in seed development is to buffer the water loss that occurs during embryo maturation and that loss of ATEM6 expression results in the mutant phenotype.