Circumventing the stability-function trade-off in an engineered FN3 domain.

Area of Science:

• Protein engineering
• Biophysical chemistry
• Structural biology

Background:

• Non-antibody protein scaffolds offer favorable biophysical properties compared to monoclonal antibodies.
• A common challenge is the stability-function trade-off, limiting gains after functional selection.
• The fibronectin Type III (FN3) domain, specifically FNfn10, previously achieved high affinity for lysozyme but lacked stability.

Purpose of the Study:

• To investigate the stability-function compromise in protein scaffolds.
• To engineer an ultra-stable FN3 scaffold with restored high binding affinity.
• To utilize structural insights for rational protein design.

Main Methods:

• Grafting lysozyme-binding loops from a high-affinity FN3 variant (FNfn10-α-lys) onto an ultra-stable FN3 scaffold (FN3con).
• Determining the crystal structure of FNfn10-α-lys in complex with lysozyme.
• Redesigning the grafted scaffold based on structural information to restore binding affinity and maintain stability.

Main Results:

• Loop grafting resulted in reduced binding affinity and retained thermal stability.
• Structural analysis revealed unanticipated framework residue interactions affecting binding.
• Redesign restored picomolar binding affinity while maintaining significantly enhanced thermodynamic stability (2-fold higher melting temperature).

Conclusions:

• Consensus design generates highly stable scaffolds suitable for protein engineering.
• The ultra-stable FN3 scaffold (FN3con) tolerates deleterious mutations, offering an advantage for functional design.
• This work demonstrates a strategy to overcome stability-function trade-offs in non-antibody protein scaffolds.