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

Area of Science:

• Biomaterials Science
• Structural Biology
• Protein Chemistry

Background:

• Liquid-liquid phase separation (LLPS) drives protein organization in intrinsically disordered proteins (IDPs) and biomaterials.
• The specific residue interactions governing LLPS-induced structural ordering in spider silk remain unclear.

Purpose of the Study:

• To elucidate the molecular role of arginine and tyrosine in *Latrodectus hesperus* dragline silk LLPS.
• To establish a mechanistic link between residue-specific chemistry, LLPS, and hierarchical assembly in structural proteins.

Main Methods:

• Isotope-edited solution NMR
• DNP-enhanced solid-state NMR
• Molecular dynamics simulations
• AlphaFold3 modeling

Main Results:

• Phosphate addition triggers LLPS, promoting Arg-Tyr cation-π interactions and weakening Arg-poly(Ala) contacts.
• Arginine integrates into β-sheet interfaces, while tyrosine adopts β-turn conformations in spun fibers.
• Arg-Tyr contacts act as critical "sticker" interactions mediating condensation and stabilizing fiber architecture.

Conclusions:

• Identified Arg-Tyr contacts as key mediators of condensation, order nucleation, and fiber stabilization in spider silk.
• Established a mechanistic link between residue chemistry, LLPS, and hierarchical assembly in structural proteins.
• Provided insights into weak multivalent interactions bridging disordered and ordered states for condensate-driven assembly and biomimetic design.