Abstract
| Original language | English |
|---|---|
| Pages (from-to) | 541-550 |
| Number of pages | 9 |
| Journal | Protein Engineering, Design and Selection |
| Volume | 29 |
| Issue number | 11 |
| DOIs | |
| Publication status | Published - 1 Nov 2016 |
Keywords
- consensus design
- loop grafting
- protein engineering
- stability-function trade-off
- X-ray crystallography
Cite this
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Circumventing the stability-function trade-off in an engineered FN3 domain. / Porebski, Benjamin T.; Conroy, Paul J.; Drinkwater, Nyssa; Schofield, Peter; Vazquez-Lombardi, Rodrigo; Hunter, Morag R.; Hoke, David E.; Christ, Daniel; McGowan, Sheena; Buckle, Ashley M.
In: Protein Engineering, Design and Selection, Vol. 29, No. 11, 01.11.2016, p. 541-550.Research output: Contribution to journal › Article › Research › peer-review
TY - JOUR
T1 - Circumventing the stability-function trade-off in an engineered FN3 domain
AU - Porebski, Benjamin T.
AU - Conroy, Paul J.
AU - Drinkwater, Nyssa
AU - Schofield, Peter
AU - Vazquez-Lombardi, Rodrigo
AU - Hunter, Morag R.
AU - Hoke, David E.
AU - Christ, Daniel
AU - McGowan, Sheena
AU - Buckle, Ashley M.
PY - 2016/11/1
Y1 - 2016/11/1
N2 - 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.
AB - 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.
KW - consensus design
KW - loop grafting
KW - protein engineering
KW - stability-function trade-off
KW - X-ray crystallography
UR - http://www.scopus.com/inward/record.url?scp=84995938487&partnerID=8YFLogxK
U2 - 10.1093/protein/gzw046
DO - 10.1093/protein/gzw046
M3 - Article
VL - 29
SP - 541
EP - 550
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
SN - 1741-0126
IS - 11
ER -