Dimerization and liquid-liquid phase separation of the nonrepetitive domains of pyriform spidroin 1 controls the pyriform silk formation

Spiders spin high performance silks with diverse mechanical properties for specific biological functions. Of these spider silk types, pyriform silk stands out as a unique combination of wet glue and dry fibers. Investigation of self-assembly process of spider silk proteins is necessary for elucidating the silk formation mechanism. However, the functions of nonrepetitive domains in the silk formation of pyriform spidroins from liquid proteins to solid fibers are still unclear, making it difficult to achieve efficient biomimetic preparations of pyriform silk with good mechanical properties. In this study, we investigate the roles of the N-linker repeat (NLR) and both terminal domains of pyriform spidroin 1 (PySp1) in the silk formation. We demonstrate for the first time that the PySp1 NLR alone is sufficient to self-assemble into high strength fibers. Moreover, we showed that the ability to promote the pyriform silk formation by the addition of the NLR. We also found that the pH-sensitive dimerization property for N-terminal domain and the liquid-liquid phase separation (LLPS) coupled with acidification triggers the self-assembly mediated by the C-terminal domain. Overall, our results provide new insight into the role of nonrepetitive domains in the pyriform silk formation mechanism and the basis for producing new protein-based materials derived from spider pyriform silk.