Diffusing protein binders to intrinsically disordered proteins.

Area of Science:

• Structural Biology
• Protein Engineering
• Biotechnology

Background:

• Intrinsically disordered proteins (IDPs) and regions (IDRs) lack stable structures, posing challenges for therapeutic and diagnostic applications.
• Developing specific and high-affinity binders for these flexible targets is crucial but lacks a general methodology.

Purpose of the Study:

• To establish a general computational approach for designing protein binders targeting intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs).
• To demonstrate the therapeutic and diagnostic potential of generated binders against specific IDPs and IDRs.

Main Methods:

• Utilized RFdiffusion, a computational protein design method, to generate binders by freely sampling target and binding protein conformations.
• Generated binders for IDPs (amylin, C-peptide, VP48, BRCA1_ARATH) and IDRs (G3BP1, IL-2RG, prion protein) with specified target conformations.
• Validated binder efficacy through in vitro dissociation constant (Kd) measurements and in-cell fluorescence imaging.

Main Results:

• Successfully generated high-affinity binders (Kd: 3–100 nM) for various IDPs and IDRs, targeting diverse conformations.
• Demonstrated cellular binding of generated binders to their respective targets.
• Showcased functional applications: G3BP1 binder disrupted stress granules, and amylin binder inhibited amyloid formation and enhanced detection.

Conclusions:

• The RFdiffusion-based approach provides a general methodology for designing binders to flexible IDPs and IDRs.
• The generated binders exhibit high affinity, specificity, and functional activity in cellular contexts.
• This method holds significant promise for developing novel therapeutics and diagnostics for diseases involving IDPs/IDRs.