Disabling spidroin N-terminal homologs' reverse reaction unveils why its intermolecular disulfide bonds have not evolved for 380 million years.

Spiders, ubiquitous predators known for their powerful silks, rely on spidroins that self-assemble from high-concentration solutions stored in silk glands, which are mediated by the NT and CT domains. CT homodimers containing intermolecular disulfide bonds enhance silk performance, promoting spider survival and reproduction. However, no NT capable of forming such disulfide bonds has been identified. Our study reveals that NT homodimers with sulfur substitution can form under alkaline conditions, shedding light on why spiders have not evolved intermolecular disulfide bonds in the NT module during their 380 million years of evolution. This discovery significantly advances our comprehension of spider evolution and silk spinning mechanisms, while also providing novel insights into protein storage, assembly, as well as the mechanisms and therapeutic strategies for neurodegenerative diseases associated with protein aggregation.