Fig. 4

Overview of the role of the N- and C-terminal domains of major ampullate spidroins during storage and fiber formation. A General scheme of the spidroin assembly process from partially dimeric to micellar assemblies and larger droplet-like condensates, which are further compressed and elongated on their way through the spinning duct. Finally, a solid silk fiber is drawn from the exit spigot. B Schematic representation of the molecular interactions during the spider silk assembly process. After secretion by epithelial cells in the tail of the upper part of the gland, the C-terminal domains (green) of the spidroins are present as parallel dimers, often further stabilized by disulfide bridges at their interfaces. Owing to hydrophobic–hydrophilic interactions, the spidroins self-assemble into micelle-like structures, with the terminal domains spanning the sphere surface, whereas the more hydrophobic repetitive core domains are oriented toward the inside of the micelle-like constructs. At this stage, the N-terminal domains (blue) remain monomeric. In the distal part of the ampulla, the micellar preassemblies undergo LLPS to form a protein-rich phase with micrometer-sized liquid-like condensates, most likely triggered by a combination of pH changes, high protein concentrations, altered viscosity, and “sticker”-like interactions of amino acid residues such as tyrosine and arginine. Strikingly, the repetitive core domain remains intrinsically unstructured under these conditions. In the spinning duct, increasing acidification to pH < 6.5 leads to antiparallel dimerization of the N-terminal domains and, thus, the formation of a crosslinked spidroin network. Moreover, the repetitive core region now becomes more structured by forming helical secondary structures. Water removal and the exchange of chaotropic ions for kosmotropic phosphate in the spinning duct induce a configurational change in the C-terminal domains, allowing for a more packed alignment of the core regions and enhanced intra- and intermolecular interactions of poly-A motifs to form β-sheet-rich crystalline regions. Owing to the elongational flow in the tapered spinning duct, shear stress stretches the preassembled silk proteins to yield the final, optimally packed and oriented nanofibrils that are pulled as solid fibers from the spigot at the end of the spinning duct. C Rendered protein structures of monomeric N- and dimeric C- and N-terminal domains (adapted structures from the RCSB PDB data bank [47,48,49]). Both the N- and C-terminal domains have a five-helix bundle structure