5J7K

Loop grafting onto a highly stable FN3 scaffold

Summary for 5J7K

• Entry DOI: 10.2210/pdb5j7k/pdb
• Related: 5J7C
• Descriptor: FN3con-a-lys, ZINC ION (3 entities in total)
• Functional Keywords: loop grafting, rational design, fibronectin type iii, fn3, protein binding, protein binding-hydrolase complex
• Biological source: synthetic construct
• Total number of polymer chains: 8
• Total formula weight: 89075.76

Authors

Porebski, B.T.,Drinkwater, N.,McGowan, S.,Buckle, A.M. (deposition date: 2016-04-06, release date: 2016-08-17, Last modification date: 2023-09-27)

Primary citation

Porebski, B.T.,Conroy, P.J.,Drinkwater, N.,Schofield, P.,Vazquez-Lombardi, R.,Hunter, M.R.,Hoke, D.E.,Christ, D.,McGowan, S.,Buckle, A.M.
Circumventing the stability-function trade-off in an engineered FN3 domain.
Protein Eng.Des.Sel., 2016

PubMed Abstract: The favorable biophysical attributes of non-antibody scaffolds make them attractive alternatives to monoclonal antibodies. However, due to the well-known stability-function trade-off, these gains tend to be marginal after functional selection. A notable example is the fibronectin Type III (FN3) domain, FNfn10, which has been previously evolved to bind lysozyme with 1 pM affinity (FNfn10-α-lys), but suffers from poor thermodynamic and kinetic stability. To explore this stability-function compromise further, we grafted the lysozyme-binding loops from FNfn10-α-lys onto our previously engineered, ultra-stable FN3 scaffold, FN3con. The resulting variant (FN3con-α-lys) bound lysozyme with a markedly reduced affinity, but retained high levels of thermal stability. The crystal structure of FNfn10-α-lys in complex with lysozyme revealed unanticipated interactions at the protein-protein interface involving framework residues of FNfn10-α-lys, thus explaining the failure to transfer binding via loop grafting. Utilizing this structural information, we redesigned FN3con-α-lys and restored picomolar binding affinity to lysozyme, while maintaining thermodynamic stability (with a thermal melting temperature 2-fold higher than that of FNfn10-α-lys). FN3con therefore provides an exceptional window of stability to tolerate deleterious mutations, resulting in a substantial advantage for functional design. This study emphasizes the utility of consensus design for the generation of highly stable scaffolds for downstream protein engineering studies.

PubMed: 27578887
DOI: 10.1093/protein/gzw046

Experimental method

X-RAY DIFFRACTION (2.46 Å)