Arg–Tyr cation–π interactions drive phase separation and β-sheet assembly in native spider dragline silk

Liquid-liquid phase separation (LLPS) is a fundamental principle of protein organization in intrinsically disordered proteins (IDPs) and biomaterials, yet the residue-level interactions that link condensation to structural ordering remain poorly defined. In spider dragline silk, LLPS is believed to initiate the transition from soluble spidroin dope into β-sheet-rich fibers that provide exceptional toughness, yet how sequence-specific motifs govern this process has been unclear. Here, we combine isotope-edited solution NMR, dynamic nuclear polarization (DNP)-enhanced solid-state NMR, molecular dynamics simulations, and AlphaFold3 modeling to define the molecular role of arginine and tyrosine in <i>Latrodectus hesperus</i> dragline silk. Phosphate triggers LLPS while preserving intrinsic disorder, with arginine exhibiting the largest chemical shift perturbations. Simulations reveal that phosphate displaces hydration water to promote Arg-Tyr cation-π interactions and weaken Arg-poly(Ala) contacts. Solid-state NMR directly detects Arg-Tyr contacts in spun fibers, demonstrating that arginine is partially incorporated into β-sheet interfaces while tyrosine frequently adopts β-turn conformations. AlphaFold3 models corroborate these interfacial geometries and reproduce experimental chemical shifts, supporting persistent Arg-Tyr interactions at structured-unstructured boundaries. Together, these results identify Arg-Tyr contacts as critical "sticker" interactions that mediate condensation, nucleate local order, and stabilize fiber architecture. More broadly, this work establishes a mechanistic link between residue-specific chemistry, LLPS, and hierarchical assembly in a structural protein. These insights highlight how weak multivalent interactions bridge disordered and ordered states, providing a general framework for condensate-driven assembly in biology and guiding biomimetic material design.