Multifunctional Defense of <i>Achelura yunnanensis</i> Cocoon: High-Strength Tough Silk Fiber, Biomineral Reinforcement, and Protease Degradation Resistance

Abstract Natural silks offer combinations of mechanical performance and biological functionality that synthetic materials still struggle to replicate. Here, we report the structural, mechanical, and biochemical characterization of cocoon silk from Achelura yunnanensis (Lepidoptera: Zygaenidae), a moth that builds a leaf-wrapped cocoon with three integrated defense layers. The silk fiber exhibits an average tensile strength of 1038 ± 434 MPa, a toughness of 81 ± 49 MJ m–3, and an elastic modulus of 22.1 ± 8.8 GPa, approximately twice the respective values for Bombyx mori silk. These properties stem from a β-sheet content of 44.0 ± 1.6% and crystallinity of 59.6%, enabled by an anterior silk gland that occupies 55% of the total gland length (vs 11.3% in B. mori) and imposes prolonged shear-driven molecular alignment during spinning. The fibroin heavy chain carries a chimeric motif architecture: silkworm-type (GAGAGSGSGA)n repeats (17.6%) coexist with (A)n segments (31.4%) and (GXGGXGXX)n motifs (14.1%) closely resembling spider dragline silk sequences. On the exposed side of the A. yunnanensis cocoon, abundant calcium oxalate monohydrate crystals (56.1% COM content) boost the specific puncture strength to 28.9 N/mm, more than double the 13.8 N/mm of B. mori. Proteomic profiling identified 36 putative antimicrobial proteins in the cocoon, dominated by trypsin inhibitor-like (TIL) proteins. Fluorescence-based enzyme inhibition assays show that these silk proteins suppress both microbial serine proteases (proteinase K residual activity: 0.7%; subtilisin: 1.8%) and animal digestive proteases (trypsin: 24.5%; chymotrypsin: 57.5%). Molecular docking of the predominant inhibitor TIL1 against all four target proteases yields binding free energies of –9.3 to –15.4 kcal/mol with extensive salt-bridge networks. A. yunnanensis cocoon silk thus functions as a naturally integrated physical–chemical composite: high-strength fibers, selective biomineral reinforcement, and broad-spectrum resistance to protease degradation, providing a model system for bioinspired material design.