C-Terminal Domains of Spider Silk Proteins Having Divergent Structures but Conserved Functional Roles

Abstract

Spider silk is self-assembled from silk proteins or spidroins. C-terminal domains (CTDs) of various types of spidroins are relatively conserved in amino acid sequences and are suggested to adopt similar structures and perform similar functional roles in spidroin storage and silk formation. Here, we solved the structure of the CTD from a capture-spiral silk protein (CTDFl) and characterized its stability and fibril formation in the presence and absence of a reducing agent at different pH values. CTDFl adopts a dimeric structure with 8 helices, but the CTDs of other types of spidroins exist in a domain-swapped dimeric structure with 10 helices. Despite the structural differences, CTDFl is pH-responsive in stability and fibril formation, similar to the CTDs from minor and major ampullate spidroins. Thus, the functional role of CTDs in silk fiber formation seems conserved. Comparing wild-type CTDFl and its mutants, we found that the pH-responsive behavior results from the protonation of H76, which is conserved from different spider species. In addition, the fibril formation rate of CTDFl correlates with its instability, suggesting that structural changes are involved in fibril formation.

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

“…The biological functions of amyloid fibril formation in the terminal domains remain unclear, but it has been experimentally confirmed in vitro that the recombinant CTs of MiSp and Flag form in a pH‐sensitive manner amyloid‐like nanofibrils that are thought to trigger the repetitive regions to rapidly form fibers, but seeding effects from these CT nanofibrils have not been reported. [ 35,36 ]…”

“…The biological functions of amyloid fibril formation in the terminal domains remain unclear, but it has been experimentally confirmed in vitro that the recombinant CTs of MiSp and Flag form in a pH-sensitive manner amyloid-like nanofibrils that are thought to trigger the repetitive regions to rapidly form fibers, but seeding effects from these CT nanofibrils have not been reported. [35,36] Given the limitations of the ArchCandy prediction, such as its inability to predict stacking of anti-parallel 𝛽-arches or 𝛽solenoidal structure, [45] we used the AMYLPRED2 consensus method to predict amyloidogenic regions in spidroins that did not exhibit 𝛽-arcade patches in ArchCandy prediction, namely the repetitive regions of Flag and MaSp1 and the NTs of MiSp and PySp. Upon AMYLPRED2 prediction, the repetitive region of MaSp1 displayed amyloid-forming regions mainly located within the poly-A motifs (Figure 2a; Figure S7, Supporting Information), supporting the notion that the poly-A motifs are responsible for forming the 𝛽-sheet nanocrystallites present in spider major ampullate silk fibers.…”

“…[31][32][33] The short dodecapeptide from the silk 𝛽sheet sequence of orb web spider Araneus ventricosus can selfassemble into 20 nm wide nanofibrils after incubation for 72 h in 5 mmol L −1 NaCl and 10 mmol L −1 phosphate buffer solution (PBS). [34] The globular N-and C-terminal domains (NT and CT) play vital roles in regulating spider silk formation, [14] in vitro experiments demonstrated that the non-repetitive CTs of A. ventricosus minor ampullate silk protein (MiSp) and flagelliform spidroin (FlSp) could spontaneously unfold and assemble into ThT-positive nanofibrils under acidic conditions, [35,36] while the recombinant NT from Euprosthenops australis MaSp1 forms nanofibril-based hydrogel. [37] While there are notable similarities between amyloid fibrils and spider silk fibrils, such as their comparable diameters, morphology, and the ability to bind amyloid-specific dyes, it is essential to acknowledge the formal definition of amyloid fibrils, which hinges upon an ordered cross-𝛽-sheet structure within the fibrillar core, leading to a fibrillar architecture.…”

Self-Assembly of RGD-Functionalized Recombinant Spider Silk Protein into Microspheres in Physiological Buffer and in the Presence of Hyaluronic Acid

“…There are, however, unsurpassed thresholds in mass-producing natural spider silk since it is challenging to farm and harvest from spiders, which is why recombinant DNA technology has been employed to develop artificial spider silk materials from key parts of the sequence of various silk proteins (spidroins). , Silk-like materials based on recombinant silk proteins successfully deliver the advantages of the natural material with additional properties, such as scalability, , biofunctionalization, − tunability, , low immune response, and improved biocompatibility. , …”

Adenylate Kinase Fused to Spidroin as a Catalyst for Decreasing Leakage out of 3D-Bioprinted Hydrogels and for ATP Regeneration

“…The excellent and diversified mechanical properties of the spider silks are granted by the repetitive numbers and composition of repeat units of the Rep domains, which could be irreversibly transformed from disordered random coil conformation to β-sheet conformation in the fiber formation process, with the change of conditions of kind and concentration of ionic liquids, pH, and shear forces . Due to their high solubility, C-terminal domains, relatively conserved in amino acid sequences, are thought to play an important role in preventing the disordered aggregation of spidroins in high concentration by remaining outside the oligomer core, formed by Rep domains in storage, and directing the ordered self-assembly of spidroins to form silk fibers . In the absence of the NT domains, which are crucial for spidroin storage in silk glands and fiber formation, truncated miniature spidroins, with only 1–4 repetitive units of Rep domains fused to CT domains, could be self-assembled to form fiber wells .…”